Heteroduplex tracking assay

ABSTRACT

A change in viral tropism occurs in many HIV positive individuals over time and may be indicated by a shift in coreceptor use from CCR5 to CXCR4. The shift in coreceptor use to CXCR4 has been shown to correlate with increased disease progression. In patients undergoing HAART, the predominant populations of virus may be shifted back to CCR5-mediated entry soon after the CXCR4-specific strains have emerged. The present invention relates to a diagnostic method to monitor coreceptor use in the treatment and clinical management of human immunodeficiency virus (HIV) infection. The present invention further relates to a diagnostic method applied to HIV-positive individuals undergoing HAART to monitor the suppression of CCR5- or CXCR4-specific strains. The diagnostic methods may be used to assist in selecting antiretroviral therapy and to improve predictions of disease prognosis over time. The methods of the invention include cell-based methods, including cell fusion assays, and molecular-based methods, including heteroduplex tracking assay, to both quantitatively and qualitatively analyze patient-derived HIV for coreceptor usage.

RELATED APPLICATIONS/PATENT'S & INCORPORATION BY REFERENCE

This application is a divisional of U.S. application Ser. No.11/654,897, filed Jan. 17, 2007, which claims priority to U.S.Provisional Application Ser. No. 60/838,009, filed Aug. 16, 2006 andwhich is a continuation-in-part of U.S. application Ser. No. 11/333,073,filed Jan. 17, 2006, and issued as U.S. Pat. No. 7,344,830, which is acontinuation-in-part of U.S. application Ser. No. 10/695,846, filed Oct.29, 2003, and issued as U.S. Pat. No. 7,294,458, which is a divisionalapplication of U.S. application Ser. No. 09/963,064, filed Sep. 25, 2001and issued as U.S. Pat. No. 6,727,060 on Apr. 27, 2004, and which claimspriority to U.S. Provisional Application Ser. No. 60/282,354, filed Apr.6, 2001 and U.S. Provisional Application Ser. No. 60/235,671, filed Sep.26, 2000.

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the PCT and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference. More generally, documents orreferences are cited in this text, either in a Reference List before theclaims, or in the text itself; and, each of these documents orreferences (“herein-cited references”), as well as each document orreference cited in each of the herein-cited references (including anymanufacturer's specifications, instructions, etc.), is hereby expresslyincorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by the government, in part, by Grant U01A135004from the National Institute for Allergy and Infectious Diseases and aNational Research Service Award (1F32HD08478-01) from the NationalInstitute of Child Health and Human Development. The government may havecertain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to a diagnostic method to monitorcoreceptor use in treatment of human immunodeficiency virus (HIV, or “anAIDS virus”) infection. This method may assist in determining when toinitiate antiretroviral therapy, in selecting antiretroviral therapy,and in predicting clinical disease progression during treatment.Moreover, the present invention relates to qualitative and quantitativemethods for evaluating patient-derived HIV samples for coreceptor use,e.g. the presence and/or absence of CCR5 and CXCR4-specific strains orshifts in coreceptor use with respect to disease progression ortreatment. The qualitative and quantitative methods of the invention mayrelate to cell-based systems, such as a cell-fusion assay, andmolecular-based systems, such as a heteroduplex tracking assay, tomonitor, measure, evaluate, detect, etc: the coreceptor use ofpatient-derived HIV. The present invention further relates to adiagnostic method to monitor the suppression of CXCR4-specific strainsin HIV infected individuals undergoing antiretroviral therapy. Thepresent invention also relates to a diagnostic method to determine HIV-1coreceptor usage and CXCR4-specific viral load to determine when toinitiate antiretroviral therapy, to predict clinical disease progressionduring combination antiretroviral therapy, and to determine when tochange therapy.

BACKGROUND OF THE INVENTION

HIV uses a receptor-mediated pathway in the infection of host cells.HIV-1 requires contact with two cell-surface receptors to gain entryinto cells and initiate infection; CD4 is the primary receptor. CXCR4and CCR5, members of the chemokine receptor family of proteins, serve assecondary coreceptors for HIV-1 isolates that are tropic for T-celllines or macrophages, respectively (Deng et al. (1996) Nature 381:661-6;Doranz et al. (1996) Cell 86:1149-59; and Berger et al. (1998) Nature391:240; Feng et al. (1996) Science 272:872-877; Samson et al. (1996)Nature 382:722-725).

CXCR4 or CCR5, in conjunction with CD4, form a functional cellularreceptor for entry of certain strains of HIV into cells. Recent reportsindicated that the viral envelope glycoprotein gp120 interacts directlywith chemokine receptors generally at a step following CD4 binding(Lapham et al. (1996) Science 274:602-605; Moore (1997) Science 276:51;Wu et al. (1996) Nature 384:179-183; and Hesselgesser et al. (1997)Current Biology 7:112-121).

Coreceptor use therefore plays a critical role in viral tropism,pathogenesis, and disease progression. HIV-1 strains transmitted in vivogenerally use CCR5 (R5 viruses), whether by sexual, parenteral, ormother-to-child transmission (Fenyo et al. (1998) Nature 391:240; Samsonet al. (1996) Nature 382:722-5; Shankarappa et al. (1999) J. Virol.73:10489-502; and Scarlatti et al. (1997); Berger et al. (1998);Bjorndal et al. (1997) J. Virol. 71:7478-7487). These viruses typicallyinfect macrophages and primary CD4+ lymphocytes, and do not formsyncytia in vitro (Journal et al. (1997) J. Virol. 71:7478-87); they aresaid to be macrophage tropic (M-tropic).

Years after chronic infection is established, strains using CXCR4 (X4strains) emerge in approximately 50% of infected individuals (Berger etal. (1998); Scarlatti et al. (1997); Koot et al. (1993); and Connor etal. (1997) J. Exp. Med. 185:621-8). This is believed to be due to theability of X4 strains to infect primary T-lymphocytes and then tofurther replicate in T-cell lines and induce syncytia (Björndal et al.(1997)); they are said to be T-cell tropic (T-tropic). X4 strains notonly infect an expanded spectrum of crucial target cells as compared toR5 viruses, but they also exhibit increased cytopathicity and mediatebystander killing of uninfected cells (Blaak et al. (2000) Proc. Natl.Acad. Sci. USA 97:1269-74; Kreisberg et al. (2001) J. Virol.75:8842-8847; Jekle et al. (2003) J. Virol. 77:5846-54).

Envelope variants selectively interact with either CXCR4 or CCR5. All ofthe known genetic determinates of coreceptor usage are found in theenvelope gene (env), with the key determinates being found in the regionof the env gene encoding the third variable (V3) domain of the gp120glycoprotein. Previously, HIV-1 coreceptor utilization had beenpredicted according to the sequence of the V3 portion of the env gene(Hung C S et al. (1999); and Briggs D R et al. (2000)). For example, anaccumulation of positively charged amino acid located in the V3 domaini.e., at positions 11 and 25 of the V3 domain and is a common feature ofX4 viruses (Fouchier R A et al. (1992); Milich L. et al. (1997)). The V3region of CXCR4-specific viruses also can exhibit greater sequencevariation than their R5-specific counterparts, in particular respectwith common laboratory HIV isolates at HTLV-IIIB/LAV and JR-CSF (MilichL. et al. (1997)).

The difference in cell tropism correlates with disease progression.Generally, after primary HIV-1 infection, viral populations are usuallycharacterized by molecular heterogeneity. Strains isolated fromindividuals early in the course of their infection are usually M-tropic(Shankarappa et al. (1999); and Glushakova et al. (1999) J. Clin.Invest. 104:R7-R11). In many cases, the X4 and R5 strains coexist tosome extent in the viral swarm or population. For example, virusesisolated from approximately 50% of individuals with advancedimmunodeficiency include viruses that are M- and T-tropic. Typically,the emergence of X4 variants is associated with depletion of CD4 cellsand acceleration of clinical disease. (See: Berger et al. (1998);Björndal et al. (1997); Shankarappa et al. (1999); Scarlatti et al.(1997); Koot et al. (1993) Ann. Intern. Med. 118:681-688; Connor et al.(1997) J. Exp. Med. 185:621-628; Blaak et al. (2000) Proc. Natl. Acad.Sci. 97:1269-1274). For example, it has been shown that cytopathicitytoward the general CD4+ T cell population in lymphoid tissue isassociated with the use of CXCR4 (Glushakova et al. (1999)).Additionally, in vitro results suggest that selective blockade of CXCR4receptors may prevent the switch from the less pathogenic R5 strains tothe more pathogenic X4 strains (Este et al. (1999) J. Virol.73:5577-85).

Current antiretroviral therapies are intended to improve the overallclinical outcome of infected individuals. For example, treatment ofinfected individuals with combination antiretroviral therapy (cART),formerly called highly active antiretroviral therapy (HAART), has led toa dramatic decline in both HIV-1-related illness and death (Palella etal. (1998) N. Engl. J. Med. 338:853-60; Egger et al. (1997) BMJ315:1194-9; Ledergerber et al. (1999) 353:863-8); Mocroft et al. (2003)362:22-9). Early clinical trials demonstrated a reduction of plasmaHIV-1 RNA loads to undetectable levels in the majority of treatedindividuals (Hammer et al. (1997) N. Engl. J. Med. 337:725-33; andAutran et al. (1997) Science 277:112-6). Subsequent studies, however,have showed more limited success. In particular although many patientsexperience initial immunologic and clinical responses to cART, thesuppression of plasma viremia is not always sustained (Deeks et al.(2000); and Mezzaroma et al. (1999)).

cART has been demonstrated to preferentially suppress X4 strains duringthe first years of therapy, suggesting that shifts in HIV-1 coreceptorusage may contribute to the clinical efficacy of treatment (Philpott etal. (2001) J. Clin. Invest. 107:431-437; Equils, et al. (2000) J.Infect. Dis. 182: 751-757; and Skrabal et al. (2003) AIDS 17:809-814).For example, in comparison to pretherapy determinations, expression ofCXCR4 was significantly increased, and CCR5 decreased, following threemonths of an anti-viral regimen; the changes in coreceptor expressionoccurred in association with a decrease in viral load and T cellactivation, and an increase in naive and memory T cells, suggestingperipheral redistribution of T cell compartments (Giovannetti et al.(1999) Clin. Exp. Immunol. 118:87-94). In another study, cART wasreported to reduce the expression of CXCR4 and CCR5 in lymphoid tissue(Andersson et al. (1998) AIDS 12:F123-9). These studies did not addresscoreceptor usage in patients undergoing HAART. The effects of cART oncoreceptor usage by viral populations were heretofore unknown.

In patients undergoing cART, the predominant populations of virus shiftback to CCR5-mediated entry after the CXCR4-specific strains emerge.cART may affect either the expression of CCR5 over CXCR4 or,alternatively, it may be influencing the kind of viral variant thatpredominates, such as CCR5-specific versus CXCR4-specific viruses. Thereis a correlation between the emergence of CXCR4-specific strains andrapid HIV disease progression.

Because cART is toxic to some patients, costly, and requires life-longadherence, the decision to start treatment in asymptomatic patients iscomplex, and therefore tailored to the individual (Yeni et al. (2004)JAMA 292:251-65). A small proportion of patients continue to experiencedisease progression despite cART, and questions remain regarding when toinitiate and switch therapies (Egger et al. (1997) Ledergerber et al.(1999); Mocroft et al. (2003); Opravil et al. (2002) AIDS 16:1371-81;Sterling et al. (2003) J. Infect. Dis. 188:1659-65); Anastos et al.(2004) Ann. Intern. Med. 140:256-64; Mezzaroma et al. (1999) Clin.Infect. Dis. 29:1423-30; Deeks et al. (2000) J. Infect. Dis. 181:946-53;and Ledergerber et al. (2004) Lancet 364:51-61). Changing therapy inthese patients, particularly after drug resistance or intolerance hasdeveloped, is also a challenge.

Currently, the principal measurements guiding therapeutic decisions areCD4 count and plasma HIV-1 RNA, as both are predictors of diseaseprogression and response to cART (Anastos et al. (2004); Yeni et al.(2004); Ledergerber et al. (2004); Kitchen et al. (2001) Clin. Infect.Dis. 33:466-72; Egger et al. (2002) Lancet 360:119-29; and Chene et al.(2003) Lancet 362:679-86). However, debate continues about optimaltreatment strategies, highlighting the need for more data to guideclinical management (Holmberg et al. (2004) Clin. Infect. Dis.39:1699-1704; Miller et al. (2002) J. Infect. Dis. 186:189-197; andPhillips et al. (2003) AIDS 17:1863-1869). In particular, new markersare necessary to identify which patients are at highest risk forclinical disease and therefore most likely to benefit from immediateinitiation or change of cART. These patients may be untreated,asymptomatic individuals or those with persistent viremia despite cART.

To accurately predict disease prognosis over time and in response totreatment, a diagnostic method would be useful to monitor the presence(or absence) of CXCR4-specific strains and/or CCR5-specific strains andshifts in coreceptor use over time. A diagnostic method for use inmonitoring shifts in coreceptor use may thereby be beneficial formeasuring the therapeutic efficacy of various HIV treatment regimes,such as cART. The effect of cART on coreceptor use by populations ofvirus has not heretofore been quantitatively studied.

The correlation between CXCR4-specific strains and rapid diseaseprogression also indicates that a diagnostic method would be useful tomonitor the presence of CXCR4-specific strains, shifts in coreceptor useassociated with HIV disease progression, and to monitor the presence ofCXCR4-specific strains and shifts in coreceptor use in patientsundergoing antiretroviral therapy.

Accordingly, diagnostic methods for use in detecting CXCR4 isolatesand/or monitoring shifts in coreceptor use (e.g. shifts fromCXCR4-specific HIV to CCR5-specific HIV and vice versa) would bebeneficial for predicting disease progression over time or in responseto treatment. Moreover, cell-based and molecular-based methods tomonitor, measure, evaluate, detect, etc. HIV coreceptor use which arereliable, accurate, and easy to use as well as being qualitative and/orquantitative in their approach would be a welcomed advance to the art.

In particular, diagnostic methods, e.g. cell-based and/ormolecular-based methods, for measuring, monitoring, evaluating,detecting, etc. patient-derived HIV samples for coreceptor usage wouldbe beneficial for evaluating HIV disease progression in the face ofvarious anti HIV treatment and therapies.

The citation or identification of any document in this application isnot an admission that such document is available as prior art.

OBJECTS AND SUMMARY OF THE INVENTION

The present invention relates to diagnostic methods and componentsthereof for determining the viral load of a population of acquiredimmunodeficiency virus using the CXCR4 coreceptor (X4-specific viralload) in a patient-derived biological sample. This invention furtherrelates to a method of determining when to initiate antiretroviraltherapy in a patient. The present invention also relates to a method ofmonitoring the efficacy of antiretroviral therapy in a patient.

The present invention encompasses a diagnostic method which may comprisedetermining the viral load of a population of acquired immunodeficiency(AIDS) virus using the CXCR4 coreceptor (X4-specific viral load) in apatient-derived biological sample comprising the steps of: (a) screeningindividual molecular clones of patient-derived acquired immunodeficiencyprimary isolate with a V3 loop sequencing assay to determine CCR5coreceptor usage and CXCR4 coreceptor usage of each individual molecularclone; (b) determining the proportion of HIV using the CCR5 coreceptor(R5) versus the CXCR4 coreceptor (X4) wherein the proportion isexpressed as a variable called the Quantity of X4 and R5 (QXR), whichrepresents the fraction of virus in a specimen using the R5 coreceptor;(c) determining coreceptor specific viral loads of the patient-derivedacquired immunodeficiency primary isolate wherein the R5-specific viralload=(VL)(QXR) and the X4-specific viral load=(VL)(1-QXR), whereinX4-specific viral load strongly predicts disease progression duringcART.

In a preferred embodiment of the method, the screening of individualmolecular clones of patient-derived acquired immunodeficiency primaryisolate to determine CCR5 coreceptor usage and CXCR4 coreceptor usage ofeach individual molecular clone is conducted with a V3 loop sequencingassay.

The present invention further encompasses a diagnostic method which maycomprise determining the viral load of a population of acquiredimmunodeficiency virus using the CXCR4 coreceptor (X4-specific viralload) in a patient-derived biological sample. In one embodiment, themethod may comprise the steps of: (a) screening individual molecularclones of patient-derived acquired immunodeficiency primary isolate witha heteroduplex tracking assay to determine the CCR5 coreceptor usage andthe CXCR4 coreceptor usage of each individual molecular clone; (b)determining the proportion of HIV using the CCR5 coreceptor (R5) versusthe CXCR4 coreceptor (X4) wherein the proportion is expressed as avariable called the Quantity of X4 and R5 (QXR), which represents thefraction of virus in a specimen using the R5 coreceptor; (c) determiningcoreceptor specific viral loads of the patient-derived acquiredimmunodeficiency primary isolate wherein the R5-specific viralload=(VL)(QXR) and the X4-specific viral load=(VL)(1−QXR), whereinX4-specific viral load strongly predicts disease progression duringcART.

In a preferred embodiment of the method if QXR=1, almost all of theviruses in the population use the R5 coreceptor; if QXR=0, almost all ofthe viruses in the population use the X4 coreceptor; and if QXR<1, theviruses in the population use a mixture of the R5 and X4 coreceptors.

Preferably, the patient-derived biological sample is any bodily fluid ortissue. In one embodiment, the biological sample may be a bodily fluidwhich may be selected from the group consisting of blood, plasma, andspinal fluid.

Preferably, the individual molecular clones may each comprise a DNAsequence corresponding to a portion of the HIV genome, the DNA sequencecomprising at least a portion of the genetic determinates of coreceptorusage.

In a preferred embodiment, the genetic determinates may be derived fromthe env gene.

In another preferred embodiment, the molecular clones each may bederived from RNA of the patient-derived HIV and correspond to the HIVgenome or a portion thereof and which comprise the genetic determinatesof coreceptor usage or a portion thereof. In another preferredembodiment, the molecular clones may be prepared by reversetranscription PCR (RT-PCR) of the RNA of the patient-derived HIV and atleast one set of oligonucleotide primers. In a more preferredembodiment, at least one set of oligonucleotide primers may consist ofthe first set of primers in Table 3. In another more preferredembodiment, at least one set of oligonucleotide primers may include asecond set of oligonucleotide primers, consisting of the second set ofprimers in Table 3. Preferably, the number of individual molecularclones may be at least 20.

In another preferred embodiment, the heteroduplex tracking assay of themethod may comprise the steps of: (a) amplifying the individualmolecular clone or a portion thereof by PCR to provide amplified DNAcomprising the genetic determinates of coreceptor usage or a portionthereof; (b) forming a population of heteroduplex molecules bycontacting the amplified DNA with a labeled probe complementary to theamplified DNA under conditions sufficient to form heteroduplexes; (c)separating the population of heteroduplex molecules using a separationmeans; (d) detecting the presence or absence of heteroduplex molecules;wherein the presence or absence of heteroduplex molecules revealscoreceptor usage. More preferably, the labeled probe may be derived froma known HIV-1 CCR5 clone or from a known HIV-1 CXCR4 clone. In anotherpreferred embodiment, the labeled probe may comprise a detectablemoiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, achemiluminescent moiety, or an enzymatic moiety.

In another preferred embodiment, the method may be used (a) to assess orpredict the degree of HIV progression, (b) to determine when to start orchange antiretroviral treatment, or (c) to monitor the efficacy ofantiretroviral treatment.

The present invention further encompasses a method of determining whento initiate antiretroviral therapy in a patient which may comprisedetermining the viral load of a population of acquired immunodeficiencyvirus using the CXCR4 coreceptor (X4-specific viral load) in apatient-derived biological sample which may comprise the steps of: (a)screening individual molecular clones of patient-derived acquiredimmunodeficiency primary isolate with a heteroduplex tracking assay todetermine the CCR5 coreceptor usage and the CXCR4 coreceptor usage ofeach individual molecular clone; (b) determining the proportion of EIIVusing the CCR5 coreceptor (R5) versus the CXCR4 coreceptor (R4) whereinthe proportion is expressed as a variable called the Quantity of X4 andR5 (QXR), which represents the fraction of virus in a specimen using theR5 coreceptor; (c) determining coreceptor specific viral loads of thepatient-derived acquired immunodeficiency primary isolate wherein theR5-specific viral load=(VL)(QXR) and the X4-specific viralload=(VL)(1−QXR), and wherein initiation or change of antiretroviraltherapy may be considered anytime that the X4-specific viral load isgreater than zero.

In a preferred embodiment, if QXR=1, almost all of the viruses in thepopulation use the R5 coreceptor; if QXR=0, almost all of the viruses inthe population use the X4 coreceptor; and if QXR<1, the viruses in thepopulation use a mixture of the R5 and X4 coreceptors.

Preferably, the patient-derived biological sample may be any bodilyfluid or tissue. In one embodiment, the biological sample may be abodily fluid which may be selected from the group consisting of blood,plasma, and spinal fluid.

Preferably, the individual molecular clones may each comprise a DNAsequence corresponding to a portion of the HIV genome, the DNA sequencecomprising at least a portion of the genetic determinates of coreceptorusage.

In a preferred embodiment, the genetic determinates may be derived fromthe env gene.

In another preferred embodiment, the molecular clones each may bederived from RNA of the patient-derived HIV and correspond to the HIVgenome or a portion thereof and which comprise the genetic determinatesof coreceptor usage or a portion thereof. In another preferredembodiment, the molecular clones may be prepared by RT-PCR of the RNA ofthe patient-derived HIV and at least one set of oligonucleotide primers.In a more preferred embodiment, at least one set of oligonucleotideprimers may consist of the first set of primers in Table 3. In anothermore preferred embodiment, at least one set of oligonucleotide primersmay include a second set of oligonucleotide primers, the second set mayconsist of the second set of primers in Table 3. Preferably, the numberof individual molecular clones may be at least 20.

In another preferred embodiment, the heteroduplex tracking assay of themethod may comprise the steps of: (a) amplifying the individualmolecular clone or a portion thereof by PCR to provide amplified DNAcomprising the genetic determinates of coreceptor usage or a portionthereof; (b) forming a population of heteroduplex molecules bycontacting the amplified DNA with a labeled probe complementary to theamplified DNA under conditions sufficient to form heteroduplexes; (c)separating the population of heteroduplex molecules using a separationmeans; (d) detecting the presence or absence of heteroduplex molecules;wherein the presence or absence of heteroduplex molecules revealscoreceptor usage. More preferably, the labeled probe may be derived froma known HIV-1 CCR5 clone or from a known HIV-1 CXCR4 clone. In anotherpreferred embodiment, the labeled probe may comprise a detectablemoiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, achemiluminescent moiety, or an enzymatic moiety.

In another preferred embodiment, the antiretroviral therapy of themethod may be any suitable antiretroviral treatment regimen. Morepreferably, the antiretroviral therapy may be selected from the groupconsisting of combination antiretroviral therapy (cART), proteaseinhibitors, fusion inhibitors, integrase inhibitors, coreceptor specificagents, nonnucleoside analogue reverse transcriptase inhibitors andnucleoside analogue reverse transcriptase inhibitors. Preferably, thenucleoside analoque reverse transcriptase inhibitor may be 3TC or AZT.Preferably, the nonnucleoside analogue reverse transcriptase inhibitormay be nevirapine.

The present invention further encompasses a method of monitoring theefficacy of antiretroviral therapy in a patient which may comprisedetermining the viral load of a population of acquired immunodeficiencyvirus using the CXCR4 coreceptor (X4-specific viral load) in apatient-derived biological sample comprising the steps of: (a) screeningindividual molecular clones of patient-derived acquired immunodeficiencyprimary isolate with a heteroduplex tracking assay to determine the CCR5coreceptor usage and the CXCR4 coreceptor usage of each individualmolecular clone; (b) determining the proportion of HIV using the CCR5coreceptor (R5) versus the CXCR4 coreceptor (R4) wherein the proportionis expressed as a variable called the Quantity of X4 and R5 (QXR), whichrepresents the fraction of virus in a specimen using the R5 coreceptor;(c) determining coreceptor specific viral loads of the patient-derivedacquired immunodeficiency primary isolate wherein the R5-specific viralload−(VL)(QXR) and the X4-specific viral load=(VL)(1−QXR), whereinX4-specific viral load strongly predicts disease progression duringcART.

In a preferred embodiment, if QXR=1, almost all of the viruses in thepopulation use the R5 coreceptor; if QXR=0, almost all of the viruses inthe population use the X4 coreceptor; and if QXR<1, a the viruses in thepopulation use a mixture of the R5 and X4 coreceptors.

Preferably, the patient-derived biological sample may be any bodilyfluid or tissue. In one embodiment, the biological sample may be abodily fluid which may be selected from the group consisting of blood,plasma, and spinal fluid.

Preferably, the individual molecular clones may each comprise a DNAsequence corresponding to a portion of the HIV genome, the DNA sequencecomprising at least a portion of the genetic determinates of coreceptorusage.

In a preferred embodiment, the genetic determinates may be derived fromthe env gene.

In another preferred embodiment, the molecular clones each may bederived from RNA of the patient-derived HIV and correspond to the HIVgenome or a portion thereof and which comprise the genetic determinatesof coreceptor usage or a portion thereof. In another preferredembodiment, the molecular clones may be prepared by RT-PCR of the RNA ofthe patient-derived HIV and at least one set of oligonucleotide primers.In a more preferred embodiment, at least one set of oligonucleotideprimers may consist of the first set of primers in Table 3. In anothermore preferred embodiment, at least one set of oligonucleotide primersmay include a second set of oligonucleotide primers, the second setconsisting of the second set of primers in Table 3. Preferably, thenumber of individual molecular clones may be at least 20.

In another preferred embodiment, the heteroduplex tracking assay of themethod may comprise the steps of: (a) amplifying the individualmolecular clone or a portion thereof by PCR to provide amplified DNAcomprising the genetic determinates of coreceptor usage or a portionthereof; (b) forming a population of heteroduplex molecules bycontacting the amplified DNA with a labeled probe complementary to theamplified DNA under conditions sufficient to form heteroduplexes; (c)separating the population of heteroduplex molecules using a separationmeans; (d) detecting the presence or absence of heteroduplex molecules;wherein the presence or absence of heteroduplex molecules revealscoreceptor usage. More preferably, the labeled probe may be derived froma known HIV-1 CCR5 clone or from a known HIV-1 CXCR4 clone. In anotherpreferred embodiment, the labeled probe may comprise a detectablemoiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, achemiluminescent moiety, or an enzymatic moiety.

In another preferred embodiment, the antiretroviral therapy of themethod may be any suitable antiretroviral treatment regimen. Morepreferably, the antiretroviral therapy may be selected from the groupconsisting of combination antiretroviral therapy (cART), proteaseinhibitors, fusion inhibitors, integrase inhibitors, coreceptor specificagents, nonnucleoside analogue reverse transcriptase inhibitors andnucleoside analogue reverse transcriptase inhibitors. Preferably, thenucleoside analoque reverse transcriptase inhibitor may be 3TC or AZT.Preferably, the nonnucleoside analogue reverse transcriptase inhibitormay be nevirapine.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE FIGURES

The following Detailed Description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may be best understood in conjunction with the accompanyingdrawings, in which:

FIG. 1 depicts the effect of combination antiretroviral therapy on HIV-1coreceptor use over time in representative study subjects. Patients 1,2, 6, 8, and 10 received new, combination therapy and Patient 13remained untreated. Arrows note the first time during the study periodthat a new combination of antiretroviral drugs was initiated. Two arrowsappear if a patient received a two drug regimen first, then HAART. Theduration of treatment with each agent is indicated. Drugs areabbreviated as follows: AZT, zidovudine; 3TC, lamivudine; Rit,ritonavir; Ind, indinavir; Saq, saquinavir; d4T, stavudine; Nel,nelfinavir; ddI, didanosine; ddC, zalcitabine; and Nev, nevirapine.

FIG. 2 depicts the dynamics of the shift in coreceptor utilizationimmediately following initiation of HAART.

FIG. 3 depicts an example of a template set-up for a PE2400 PCRtray-retainer.

FIG. 4 depicts an example of a pattern produced by gel analysis based onan original RT layout, for use in selecting samples to becloned/sequenced.

FIG. 5 provides a graphical illustration of the various steps of theheteroduplex tracking assay (HTA) of the invention which provides forboth qualitative and quantitative analysis of HIV coreceptor usage.

FIG. 6 provides a schematic representation of heteroduplex trackingassay (HTA) analysis of four different targets, including probe only,CCR5-specific HIV V3 region only, CXCR4-specific HIV V3 region only, anda mixture or “quasispecies” of both CCR5-specific and CXCR4-specific HIVV3 regions.

FIG. 7 provides Kaplan-Meier curves of association of clinicalprogression with baseline QXR (QXR=1 vs. QXR<1).

DETAILED DESCRIPTION

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology, genetic engineering, polypeptide and nucleicacid synthesis, nucleic acid sequencing, cloning technology, protein/DNAexpression technology, and immunology, which are all within the skill ofthe art. Such techniques are explained fully in the literature. Seee.g., Sambrook, et al., MOLECULAR CLONING; A LABORATORY MANUAL, SECONDEDITION (1989); DNA CLONING, VOLUMES I AND II (D. N Glover ed. 1985);OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed, 1984); NUCLEIC ACIDHYBRIDIZATION (B. D. Hames & S. J. Higgins eds. 1984); TRANSCRIPTION ANDTRANSLATION (B. D. Hames & S. J. Higgins eds. 1984); ANIMAL CELL CULTURE(R. I. Freshney ed. 1986); IMMOBILIZED CELLS AND ENZYMES (IRL Press,1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); theseries, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFERVECTORS FOR MAMMALIAN CELLS (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol.155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker,eds. (1987), IMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY(Academic Press, London), Scopes, (1987), PROTEIN PURIFICATION:PRINCIPLES AND PRACTICE, Second Edition (Springer-Verlag, N.Y.), andHANDBOOK OF EXPERIMENTAL IMMUNOLOGY, VOLUMES I-IV (D. M. Weir and C. C.Blackwell eds 1986), each of which are incorporated herein by reference.

The term “or (a) fragment(s) thereof” as employed in the presentinvention and in context with polypeptides of the invention, comprisesspecific peptides, amino acid stretches of the polypeptides as disclosedherein. It is preferred that said “fragment(s) thereof” is/arefunctional fragment(s). The term “functional fragment” as used hereindenotes a part of the above identified polypeptide of the inventionwhich fullfils, at least in part, physiologically and/or structurallyrelated activities of the polypeptide of the invention. It is alsoenvisaged that the fragments, like the full-length polypeptides, may bedistinguished between HIV strains in effecting binding. The polypeptidesof the present invention can be recombinant polypeptides expressed ineukaryotic cells, like mammalian cells.

The term “nucleic acid hybridization” may be used herein to refer to“molecular-based assays,” and may include, for example, the heteroduplexbinding assay of the invention. The present invention may also includemethods that combine both cell-based and molecular based methods andshould not be construed to be limited to either one or the otherapproach.

The term “molecular clone” may be used herein to refer to the cloning ofa portion of the HIV genome, such as a gene or a portion of a gene,which can then be analyzed in accordance with the molecular-basedmethods of the invention, especially the heteroduplex tracking assay.

The term “genetic determinates” may be used herein to refer to themolecular clones of portions of the env gene which allow a quantitativedetermination of the proportion of HIV specific for the CCR5 coreceptorand those specific for the CXCR4 coreceptor, for example the thirdvariable (V3) region of the gp120 glycoprotein.

The term “PCR” as used herein refers to the molecular biology techniqueknown as polymerase chain reaction, disclosed by Mullis in U.S. Pat.Nos. 4,683,195 (Mullis et al) and 4,683,202, incorporated herein byreference. The following U.S. Patents may also be referenced forinformation relating to PCR generally: 6,316,192; 6,309,837; 6,300,073;6,300,072; 6,284,455; 6,270,977; 6,270,966; 6,268,143; 6,261,431;6,251,607; 6,232,079; 6,225,093; 6,218,153; 6,207,425; 6,183,963;6,180,372; 6,146,834; 6,087,097; 6,072,369; 6,068,974; 6,063,563;6,046,039; 6,031,960; 6,017,699; 6,015,664; 6,015,534; 6,001,612;5,972,602; 5,909,468; 5,905,732; 5,888,740; 5,883,924; 5,869,318;5,853,991; 5,837,468; 5,827,657; 5,824,516; 5,824,479; 5,814,489;5,780,222; 5,776,686; 5,774,497; 5,759,822; 5,716,784; 5,712,125;5,712,090; 5,691,146; 5,681,741; 5,618,703; 5,618,702; 5,565,340;5,556,774; 5,556,773; 5,527,510; 5,487,993; 5,426,026; 5,393,657;5,364,790; 5,364,758; 5,229,297; and 5,187,060; each of which areincorporated herein in their entirety by reference. The term RT-PCRrefers to reverse transcription of an RNA molecule to a complementaryDNA (cDNA) molecule, followed by PCR of that cDNA.

The term “patient” as used herein may be any animal, preferably amammal, and even more preferably a human, infected with HIV.

The term “acquired immunodeficiency virus” as used herein refers to theinfectious AIDS virus known to one of skill in the art and may be, butis not limited to, HIV-1 and/or HIV-2.

The term “genotype” may be used herein to refer to a strain of HIV atthe genetic sequence level. One of skill in the art appreciates thatduring the course of disease progression the pool of HIV in an infectedindividual may become a mixture of different strains which are differentat the genetic level (i.e. have different “genotypes”). It is furtherunderstood by the skilled person that whether any particular strain ofHIV from a population of virus in an infected individual is specific forCCR5 coreceptor or the CXCR4 coreceptor is dependent on the geneticdeterminates contained in that virus's genome, i.e. is reflected in thatvirus's genotype.

The term “HAART” as used herein refers to any highly activeantiretroviral therapy and is more recently referred to as combinationantiretroviral therapy, or “cART”, used interchangeably herein with“CART”. HAART and cART are also used herein interchangeably. HAART mayrefer to three or more antiretroviral drugs in combination, and usuallycomprises one protease inhibitor and two or three reverse transcriptaseinhibitors.

Methods for sequencing and/or identifying the V3 region may be anydesired method, e.g., a method which is by or analogous to the methodscited in U.S. Pat. Nos. 7,160,992; 7,157,225;7,122,646; 7,118,874;7,118,751; 7,097,970; 7,097,965; 7,090,848; 7,067,117; 7,063,943;7,063,849; 7,041,441; 7,037,896; 7,030,234; 7,022,814; 7,018,835;7,018,633; 6,995,008; 6,989,435; 6,974,866; 6,964,763; 6,955,900;6,942,852; 6,930,174; 6,926,898; 6,923,970; 6,919,319; 6,916,605;6,908,734; 6,908,617; 6,908,612; 6,897,301; 6,887,977; 6,884,623;6,881,828; 6,875,737; 6,869,925; 6,855,804; 6,855,539; 6,855,528;6,855,321; 6,849,261; 6,821,955; 6,812,026; 6,808,877; 6,806,079;6,806,055; 6,800,447; 6,797,811; 6,773,915; 6,740,747; 6,740,525;6,737,521; 6,737,267; 6,727,060; 6,713,286; 6,709,828; 6,696,289;6,692,938; 6,686,333; 6,660,271; 6,649,735; 6,649,409; 6,627,197;6,623,940; 6,613,563; 6,610,542; 6,602,705; 6,600,012; 6,596,279;6,592,872; 6,569,418; 6,562,347; 6,551,824; 6,548,636; 6,548,635;6,548,631; 6,544,752; 6,544,527; 6,534,312; 6,531,587; 6,531,137;6,528,626; 6,525,173; 6,521,739; 6,518,030; 6,511,801; 6,509,018;6,506,554; 6,503,732; 6,493,637; 6,492,123; 6,492,110; 6,482,919;6,475,492; 6,455,314; 6,451,322; 6,451,313; 6,448,375; 6,448,070;6,432,675; 6,428,970; 6,420,545; 6,410,326; 6,410,318; 6,399,294;6,395,275; 6,392,029; 6,372,425; 6,355,785; 6,355,247; 6,342,228;6,331,404; 6,329,202; 6,329,147; 6,323,185; 6,319,503; 6,303,292;6,294,654; 6,291,650; 6,291,157; 6,288,042; 6,277,561; 6,261,558;6,258,932; 6,235,714; 6,225,447; 6,214,540; 6,187,748; 6,187,310;6,177,549; 6,172,197; 6,168,784; 6,162,631; 6,156,541; 6,143,876;6,133,029; 6,132,992; 6,120,992; 6,114,115; 6,110,465; 6,080,408;6,060,064; 6,057,102; 6,042,832; 6,034,223; 6,025,125; 6,020,468;6,017,880; 6,015,661; 6,010,895; 5,980,899; 5,977,318; 5,969,109;5,969,108; 5,968,815; 5,968,510; 5,965,532; 5,962,311; 5,955,647;5,955,342; 5,925,7411; 5,919,462; 5,912,338; 5,889,176; 5,885,796;5,885,580; 5,885,579; 5,879,925; 5,871,907; 5,866,320; 5,866,137;5,863,542; 5,858,657; 5,858,366; 5,856,185; 5,852,186; 5,851,795;5,849,475; 5,844,095; 5,843,634; 5,840,480; 5,840,300; 5,837,242;5,827,666; 5,817,316; 5,807,979; 5,804,440; 5,798,205; 5,786,199;5,770,427; 5,766,845; 5,766,599; 5,766,598; 5,763,574; 5,762,938;5,759,770; 5,756,674; 5,756,312; 5,756,103; 5,744,144; 5,733,760;5,728,520; 5,714,374; 5,693,752; 5,693,325; 5,670,153; 5,670,152;5,667,782; 5,658,779; 5,652,144; 5,652,138; 5,637,481; 5,607,847;5,591,823; 5,580,773; 5,565,332; 5,541,100; 5,534,257; 5,494,807;5,443,828.

The sequence variation of the V3 loop may be detected by performing anynucleic acid analysis techniques known to those of skill in the art.Some examples of suitable techniques include sequencing techniques(direct DNA sequencing which is also known as population-basedsequencing (using either the dideoxy chain termination method or theMaxam-Gilbert method (see Sambrook et al., Molecular Cloning, ALaboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al.,Recombinant DNA Laboratory Manual, (Acad. Press, 1988)), sequencing ofsingle variants), pyrosequencing, gel electrophoresis sequencing),hybridization (heteroduplex tracking assay, line probe assay, nucleicacid arrays (details on the use of nucleic acid arrays (DNA chips) forthe detection of, for example, SNPs, see U.S. Pat. No. 6,300,063 issuedto Lipshultz, et al., and U.S. Pat. No. 5,837,832 to Chee, et al.), beadarray).

Other nucleic acid analysis techniques include restriction fragmentlength polymorphism analysis, cleavase fragment length polymorphismanalysis as described in U.S. Pat. No. 5,843,669, random amplifiedpolymorphic DNA (RAPD) analysis, arbitrary fragment length polymorphisms(AFLPs), differential sequencing with mass spectrometry, single basedextension (SBE) of a fluorescently-labeled primer coupled withfluorescence resonance energy transfer (FRE™) between the label of theadded base and the label of the primer as described by Chen et al.,(PNAS 94:10756-61 (1997), single-strand conformation polymorphismanalysis as described in Orita et al., Proc. Nat. Acad. Sci. 86,2766-2770 (1989), denaturing gradient gel electrophoresis whereinamplification products generated using PCR can be analyzed by the use ofdenaturing gradient gel electrophoresis based on the differentsequence-dependent melting properties and electrophoretic migration ofDNA in solution. Erlich, ed., PCR Technology. Principles andApplications for DNA Amplification, (W.H. Freeman and Co, New York,1992), Chapter 7.

Optionally, high throughput analysis may be achieved by PCR multiplexingtechniques well known in the art. (E.g., Z. Lin et al., Multiplexgenotype determination at a large number of gene loci, Proc. Natl. Acad.Sci. USA 93(6):2582-87 [1996]).

Optionally, additional methodologies may be achieved by combiningexisting nucleic acid analysis methodologies. An example is ultradeepsequencing wherein a two stage PCR technique coupled with a novelpyrophosphate sequencing technique would allow the detection of sequencevariants (SNP, indels and other DNA polymorphisms) in a rapid, reliable,and cost effective manner.

Conformation-sensitive gel electrophoresis of amplification products mayalso be used to analyze sequence variation of the V3 loop. (A. Markoffet al., Comparison of conformation-sensitive gel electrophoresis andsingle strand conformation polymorphism analysis for detection ofmutations in the BRCA1 gene using optimized conformation analysisprotocols, Eur. J. Genet. 6(2):145-50 [1998]). The sequence variation ofthe V3 loop may also be detected by performing immunological analysistechniques known to those of skill in the art such as ELISA and proteinarrays. The structure of the V3 loop helps to determine HIV coreceptorusage, and therefore methods that characterize V3 structure may also beused to determine whether a viral variant uses CCR5 or CXCR4 (T. Cardozoet al, Structural basis for coreceptor selectivity by the HIV-1 V3 loop.2007 AIDS Res and Hum Retroviruses; in press).

One ordinarily skilled in the art would acknowledge that there are anumber of additional methods that may be employed for analyzing sequencevariation aside from the preferred methods described herein. The presentinvention encompasses the following non-limiting types of sequencevariation analysis assays: PCR-free genotyping methods, single-stephomogeneous methods, homogeneous detection with fluorescencepolarization, “Tag” based DNA chip system, fluorescent dye chemistry,TaqMan genotype assays, Invader genotype assays, and microfluidicgenotype assays, among others.

The authors of the present invention have surprisingly found that theviral load of acquired immunodeficiency virus in a patient-derivedbiological sample using the CXCR4 coreceptor (X4-specific viral load) isdirectly related to disease progression and clinical outcome. The datapresented herein strongly suggest that the X4-specific viral loaddetermined by the methods provided herein is a powerful predictor inguiding clinical therapies including when to initiate antiretroviraltherapy, the response to antiretroviral therapies, and clinicalmanagement.

The present invention relates to diagnostic methods and componentsthereof for determining the viral load of a population of acquiredimmunodeficiency virus using the CXCR4 coreceptor in a patient-derivedbiological sample. The invention further relates to a method ofdetermining when to initiate antiretroviral therapy in a patient. Thepresent invention also relates to a method of monitoring the efficacy ofantiretroviral therapy in a patient.

The present invention encompasses a diagnostic method which may comprisedetermining the viral load of a population of acquired immunodeficiencyvirus using the CXCR4 coreceptor (X4-specific viral load) in apatient-derived biological sample. In one embodiment, the methodcomprises the steps of: (a) screening individual molecular clones ofpatient-derived acquired immunodeficiency primary isolate with aheteroduplex tracking assay to determine the CCR5 coreceptor usage andthe CXCR4 coreceptor usage of each individual molecular clone; (b)determining the proportion of HIV using the CCR5 coreceptor (R5) versusthe CXCR4 coreceptor (R4) wherein the proportion is expressed as avariable called the Quantity of X4 and R5 (QXR), which represents thefraction of virus in a specimen using the R5 coreceptor; (c) determiningcoreceptor specific viral loads of the patient-derived acquiredimmunodeficiency primary isolate wherein the R5-specific viralload+(VL)(QXR) and the X4-specific viral load=(VL)(1-QXR).

In a preferred embodiment of the method if QXR=1, almost all of theviruses in the population use the R5 coreceptor; if QXR=0, almost all ofthe viruses in the population use the X4 coreceptor; and if QXR<1, theviruses in the population use a mixture of the R5 and X4 coreceptors.

Preferably, the patient-derived biological sample is any bodily fluid ortissue. In one embodiment, the biological sample may be a bodily fluidwhich may be selected from the group consisting of blood, plasma, andspinal fluid. In another embodiment, the biological sample may be onewhich contains viral populations that are distinct from those in thereadily obtained peripheral blood including the reservoirs of thegenital tract and lymphoid tissue.

Patient-derived biological samples may be obtained by methods known toone of skill in the art. For instance, peripheral blood of HIV-infectedindividuals can be separated into plasma and cell components by methodsknown in the art. Primary viral isolates of HIV-1 may also be obtainedby co-culture with normal donor peripheral blood mononuclear cells(PBMCs). Titration of viral isolates in PBMCs can be carried out. Thesestandard techniques are described throughout the literature; forexample, see Fang et al. (1995) Proc. Natl. Acad. Sci. USA 92:12110-4

Preferably, the individual PCR products or molecular clones eachcomprise a DNA sequence corresponding to a portion of the HIV genome,the DNA sequence comprising at least a portion of the geneticdeterminates of coreceptor usage.

In a preferred embodiment, the genetic determinates are derived from theenv gene. The envelope protein may comprise gp120, gp 160 or a portionthereof. Envelope sequences are predictive of coreceptor use on thebasis of the overall charge of the V3 loop and the presence of basic oracidic residues at positions 275 and 287 of the env gene (Bhattacharyaet al. (1996) AIDS Res. Hum. Retrovir. 12:83-90; Hung et al. (1999) J.Virol. 73:8216-26); and Cardozo et al. (2007) AIDS Res. Hum. Retrov., Inpress.

Cloning strategies for isolating envelope genes of interest are wellknown to one of skill in the art. See, for example, Sambrook, Fritschand Maniatis, Molecular Cloning, A Laboratory Manual, 2^(nd) Ed., ColdSpring Harbor Laboratory Press, 1989.

Preferably, the cloning methods used in the present invention willdecrease the chance of sampling error or recombination. For example,high fidelity cloning of the samples above may be achieved by routineperformance of multiple long RT-PCR reactions on limiting dilutions ofRNA, followed by multiple PCR's on cDNAs obtained from each RT reaction.In addition, performance of multiple PCR's on each cDNA preparationincreases the likelihood of amplifying a different HIV-1 RNA species.Short-term limited dilution techniques are also well known to one ofskill in the art, see for example, Connor et al. (1997). Furthermore,quantitation of HIV-1 RNA in the biological samples of the methodsdescribed herein may be carried out, for example, by using NucliSens(Organon Teknika Corp., Durham, N.C.). Quantitation methods may setouter limits. In a preferred embodiment, RNA is amplified to ≦80copies/ml.

In a preferred embodiment of the invention, the molecular clones eachare derived from RNA of the patient-derived HIV and correspond to theHIV genome or a portion thereof and which comprise the geneticdeterminates of coreceptor usage or a portion thereof. In anotherpreferred embodiment, the molecular clones are prepared by RT-PCR of theRNA of the patient-derived HIV and at least one set of oligonucleotideprimers. In a more preferred embodiment, at least one set ofoligonucleotide primers consists of the first set of primers in Table 3.In another more preferred embodiment, at least one set ofoligonucleotide primers includes a second set of oligonucleotideprimers, the second set consisting of the second set of primers in Table3. Preferably, the number of individual molecular clones is at least 20.

In a preferred embodiment, the heteroduplex tracking assay of the methodmay comprise the steps of: (a) amplifying the individual molecular cloneor a portion thereof by PCR to provide amplified DNA comprising thegenetic determinates of coreceptor usage or a portion thereof; (b)forming a population of heteroduplex molecules by contacting theamplified DNA with a labeled probe complementary to the amplified DNAunder conditions sufficient to form heteroduplexes; (c) separating thepopulation of heteroduplex molecules using a separation means; (d)detecting the presence or absence of heteroduplex molecules; wherein thepresence or absence of heteroduplex molecules reveals particularcoreceptor usage. More preferably, the labeled probe may be derived froma known HIV-1 CCR5 clone or from a known HIV-1 CXCR4 clone. In anotherpreferred embodiment, the labeled probe comprises a detectable moiety, aradioisotope, biotin, a fluorescent moiety, a fluorophore, achemiluminescent moiety, or an enzymatic moiety.

The heteroduplex tracking assay of the invention can be carried outsubstantially in accordance with the guidance of Delwart et al. (J.Virol. (1994) 68:6672-6683), Delwart et al. (Science (1993)262:1257-1261), Nelson et al. (J. Virol. (1997) 71:8850-8, Delwart etal. (PCR Methods and Applications 4:S202-S216 (19950 Cold SpringsHarbor), and U.S. Pat. No. 5,851,759 (Weiner), each of which areincorporated in their entireties by reference.

The heteroduplex tracking assay can be used to analyze a portion of theHIV-1 genome encompassing determinates of coreceptor utilization tounderstand, determine, monitor, or detect coreceptor usage. Geneticdeterminates of HIV-1 coreceptor utilization can be found in theenvelope gene (env), with key determinates being found in the thirdvariable (V3) domain of the gp120 glycoprotein.

The heteroduplex tracking assay of the invention can be carried outgenerally, while not being limited thereto, according to the basic stepsof: (a) obtaining HIV viral RNA from the patient, (b) amplifying, e.g.PCR and/or reverse transcription (RT-PCR), a portion of the viral genomecontaining genetic determinates of coreceptor usage, e.g. a genomicportion comprising the V3 domain of the gp120 envelope glycoprotein, (c)forming heteroduplexes and/or homoduplexes with labeled nucleicacid-based probes prepared from a corresponding genomic region of aknown HIV strain, e.g. the same genomic portion comprising the V3 domainof gp120, and (d) subjecting the heteroduplexes and homoduplexes to aseparation system, e.g. electrophoresis through non-denaturingpolyacrylamide gels, wherein the heteroduplexes and homoduplexes havediffering and distinguishable mobilities that results in differentmobility patterns, e.g. a electrophoretic pattern, such that thecoreceptor usage can be determined.

For example, the presence of an electrophoretic pattern characteristicof X4-heteroduplexes can indicate the presence of CXCR4-specific virusesin the HIV sample. Alternately, the presence of an electrophoreticpattern characteristic of homoduplexes and R5-heteroduplexes canindicate the presence of only CCR5-specific viruses. And, a patterncharacteristic of both homoduplexes and X4- and R5-heteroduplexes canindicate that the HIV sample contains a mixed population ofCCR5-specific and CXCR4-specific viruses. The heteroduplex trackingassay can be performed at any point during disease progression orduring, before, or after administering antiretroviral therapy. Further,the heteroduplex tracking assay can be carried out either to attainqualitative results or quantitative results.

Methods for obtaining and/or extracting HIV RNA from patient-derivedsamples are well-known. Also, the step of amplifying a portion of theviral genome containing genetic determinates of coreceptor usage a knownin the art, and include, for example reverse transcription PCR (RT-PCR).Following RT-PCT, further rounds of PCR can be used to further amplifydesired portions of the genome, especially regions containing geneticdeterminates of coreceptor usage.

It will be appreciated that the heteroduplex tracking assay is based onthe observation that when sequences were amplified by nested PCR fromperipheral blood mononuclear cells of infected individuals, related DNAproducts coamplified from divergent templates could randomly reanneal toform heteroduplexes that migrate with reduced mobility in neutralpolyacrylamide gels. Using these techniques, one can establish geneticrelationships between multiple viral DNA template molecules, such as thedifferent genetic types (i.e. different genotypes) of HIV utilizing thedifferent coreceptors. The HTA of the invention can be described asutilizing a first PCR product as a labeled probe, e.g. radioactive, ornonradioactive which is mixed with an excess (“driver”) of an unlabeledPCR product from a different source, i.e., the source for which typingor analysis of is desired, e.g. the PCR product defining the portion ofthe HIV genome with the coreceptor genetic determinates. The probesequences are then “driven” completely into heteroduplexes with thedriver, and are separated, e.g. by gel electrophoresis, on the basis ofsize. An autoradiogram or fluoroimage, for example, of the resultingpolyacrylamide gel reveals these heteroduplexes and provides a visualdisplay of the relationship between the two virus populations understudy. The fact that heteroduplexes migrate with distinct mobilitiesindicates that the strand-specific composition of mismatched andunpaired nucleotides affects their mobility.

A “heteroduplex” encompasses a double stranded DNA molecule havingcomplementary strands at which one strand (the “target strand”, i.e. asingle strand of DNA from the PCR product of the HIV genome) containsone or more mismatched or an unpaired nucleotide base. For example, aheteroduplex can form by mixing together a labeled probe (e.g. adouble-stranded DNA PCR product of a portion of the env gene ofCCR5-specific HIV) and a PCR product of a target sequence (e.g. adouble-stranded DNA PCR product of the corresponding portion of the envgene of a CXCR4-specific HIV) such that complementary single-strandedDNA of each PCR product are combined together as a new, double-strandedmolecule. However, since the PCR product from the CXCR4-specific HIVwill contain genetic determinates characteristic of CXCR4 type viruses,its nucleotide sequence will vary at specific locations with respect tothe probe PCR product (which is derived from CCR5). These differences insequence result in a heteroduplex which has reduced mobility duringelectrophoresis with respect to homoduplexes oweing to a reduced levelof base-pairing in the molecule. In contrast, the “homoduplex” may beformed between complementary strand pairs derived from a probe PCRproduct and a target PCR product such that their nucleotide sequencesare the same.

In another embodiment, the heteroduplex tracking assay can comprise thesteps of (a) amplifying an individual molecular clone or a portionthereof by PCR to provide amplified DNA comprising the geneticdeterminates of coreceptor usage or a portion thereof; (b) forming apopulation of heteroduplex molecules by contacting the amplified DNAwith a labeled probe complementary to the amplified DNA under conditionssufficient to form heteroduplexes; (c) separating the population ofheteroduplex molecules using a separation means: and (d) detecting thepresence or absence of heteroduplex molecules; wherein the presence orabsence of heteroduplex molecules reveals coreceptor usage. In oneembodiment, the labeled probe may be derived from a known HIV-1 CCR5clone. In another embodiment, the labeled probe may be derived from aknown HIV-1 CXCR4 clone. The labeled probe can comprise a detectablemoiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, achemiluminescent moiety, or an enzymatic moiety. Appropriate labels andtheir methods of preparation are well-known.

It is furthermore envisaged that the diagnostic method may involve theuse of micro-chips comprising nucleic acid molecules encoding a envelopeprotein, or a fragment thereof, preferably a V3 region fragment,especially including genetic determinates of coreceptor usage, on genechips; or an envelope protein, or a fragment thereof, preferably a V3region fragment, on protein-chips (See U.S. Pat. Nos. 6,066,454;6,045,996; 6,043,080; 6,040,193; 6,040,138; 6,033,860; 6,033,850;6,025,601; 6,022,963; 6,013,440; 5,968,740; 5,925,525; 5,922,591;5,919,523; 5,889,165; 5,885,837; 5,874,219; 5,858,659; 5,856,174;5,856,101; 5,843,655; 5,837,832; 5,834,758; 5,831,070; 5,770,722;5,770,456; 5,753,788; 5,744,305; 5,733,729; 5,710,000; 5,631,734;5,599,695; 5,593,839; 5,578,832; and 5,556,752). Diagnostic gene chipsmay comprise a collection of polypeptides that specifically detect aenvelope protein, or fragments thereof, preferably V3 region fragments;or nucleic acid molecules that specifically detect a nucleic acidmolecule encoding a envelope protein, or fragments thereof, preferablyV3 region fragments; all of which may be used for the purposes ofdetermining coreceptor use. The envelope protein may be gp160, gp120, ora portion thereof.

It will be understood that the heteroduplex tracking assay of theinvention can be used to provide both qualitative and quantitativeinformation. First, qualitative information can be derived using the HTAof the invention by analyzing the whole HIV population derived from aninfected patient to determine whether the isolated population of HIV isCCR5-specific, CXCR4-specific, or mixture of both types. It will beappreciated that qualitative information is based on the whole orsubstantially the whole HIV population rather than individual clonestherefrom. On the contrary, quantitative information can be derivedusing the HTA of the present invention by analyzing individual HIVclones (e.g. cloned portions of the HIV genome of a plurality ofindividual HIV viruses from the isolated whole population of HIV fromthe infected patient) with respect to their coreceptor usage anddetermining a ratio of CCR5-specific to CXCR4-specific clones. In oneembodiment, the invention relates to determining the QXR ratio: thenumber of HIV clones that are identified as CCR5-specific compared tothe total number of clones analyzed.

It will be appreciated that the HIV clone can refer to the cloned PCRproduct. The quantitative HTA is performed by using clones. Aqualitative HTA is performed before a quantitiative HTA is done; thequalitative HTA is performed on the PCR-amplified portion of the HIVgenome and which contains genetic determinates of the coreceptorpreference. A qualitative HTA yields a QXR with a result of QXR=1, orQXR<1; a quantitative HTA provides a numerical measure of QXR whenQXR<1.

FIG. 5 depicts a flow chart showing the qualitative and quantitativeaspects of the HTA of the present invention. First, HIV RNA is extractedfrom the infected patient. Next, RT-PCR is carried out to obtain HIVcDNA, from which a PCR product (i.e. PCR amplicon) containing geneticdeterminates for coreceptor usage is amplied using PCR. The PCR productis then gel purified. Presumably, the PCR product will be a mixedpopulation of molecules—those genotypic for either CCR5 or CXCR4coreceptors—whenever the isolated HIV sample contains both types ofviruses. Next, the PCR product is analyzed by the HTA of the invention,which includes generally the steps of mixing together a labeled probe(e.g. a PCR product corresponding to same region in a known CCR5 strainas the amplified target PCR amplicon to be analyzed) and the amplifiedtarget PCR amplicon to form homo- or heteroduplexes. The molecules arethen separated by gel electrophoresis, for example, on a 12%polyacrylamide gel. Electrophetic techniques are well known in the art.If the QXR<1 on the qualitative test, then a quantitative test can bedone. To perform the quantitative test, the V3 portion of the HIVenvelope gene is molecularly cloned and each of 20 clones is analyzed byan individual HTA.

Exemplarly results are represented in FIG. 6. The figures shows fourpanels of schematic electropherograms. The first panel is the negativecontrol, i.e. labeled probe only. The second panel shows the result ofHTA of the V3 portion of the HIV envelope gene of a CCR5 virus. Thethird panel shows the result of HTA of the V3 portion of the HIVenvelope gene of a CXCR4 virus. And, the fourth panel shows the resultof HTA of a mixture of CCR5 and CXCR4 virus V3 regions. Four differentprobes (each based on a CCR5-specific control virus) were used to testeach HIV sample. The gels show heteroduplex band patterns for those HIVsamples containing CXCR4-specific and CCR5-specific viruses.

The quantitative results of the heteroduplex tracking assay of themethod of the present invention may be assessed by statistical methodswell known to one of skill in the art. For example, QXR, the proportionof plasma HIV-1 using CCR5, may be stratified into two categories: QXR=1if all virus identified uses CCR5, and QXR<1 if X4 virus is detected.The association between virologic responses and baseline QXR may beassessed by comparing the percentages of patients with undetectableHIV-1 RNA load across the different strata by using, for example,Fischer's exact test. Further, immunologic responses across two stratamay be compared by Wilcoxon rank-sum tests. Kaplan-Meier curves and Coxproportional hazard regression models may be applied to quantify theassociation of baseline or follow-up QXR (equal 1 versus less than 1)with subsequent clinical progression, defined as a new clinicalAIDS-defining event or death.

The quantitative results of the heteroduplex tracking assay of themethod of the present invention may further be assessed by otherstatistical methods well known to one of skill in the art. For example,an additional model analyzing the relationship of X4 viral load to HIV-1disease progression may be included by stratifying the X4-specific viralload into three strata: patients without detectable X4-specific viralload (i.e., QXR=1) and patients with detectable X4 viraemia below andabove the median value of X4-specific viral loads, respectively. Tocompare the predictive capacity with the established progression markersCD4 and HIV-1 RNA load the concurrent log₂ transformed CD4 values andlog₁₀ transformed HIV-1 loads in the univariable and multivariable Coxmodels may be included. Further, the inverse probability weights may beused to adjust for sampling bias.

Preferably, STATA (Version 9.1, StataCorp, College Station, Texas) maybe used for quantitative analyses.

In another preferred embodiment, the method is used (a) to assess orpredict the degree of HIV progression, (b) to determine when to start orchange antiretroviral treatment, or (c) to monitor the efficacy ofantiretroviral treatment. One of skill in the art (e.g. a physician,preferably one specializing in the treatment of infectious disease)would use appropriate judgment and discretion in determining how oftento apply the diagnostic methods for a patient. The frequency ofapplication may vary, depending on various factors, for example, theage, sex, type of antiretroviral therapy administered to, or stage ofdisease progression in, a patient.

The present invention further encompasses a method of determining whento initiate antiretroviral therapy in a patient which may comprisedetermining the viral load of a population of acquired immunodeficiencyvirus using the CXCR4 coreceptor (X4-specific viral load) in apatient-derived biological sample comprising the steps of: (a) screeningindividual molecular clones of patient-derived acquired immunodeficiencyprimary isolate with a heteroduplex tracking assay to determine the CCR5coreceptor usage and the CXCR4 coreceptor usage of each individualmolecular clone; (b) determining the proportion of HIV using the CCR5coreceptor (R5) versus the CXCR4 coreceptor (R4) wherein the proportionis expressed as a variable called the Quantity of X4 and R5 (QXR), whichrepresents the fraction of virus in a specimen using the R5 coreceptor;(c) determining coreceptor specific viral loads of the patient-derivedacquired immunodeficiency primary isolate wherein the R5-specific viralload=(VL)(QXR) and the X4-specific viral load=(VL)(1−QXR) and whereininitiation or change of antiretroviral therapy may be considered anytimethat the X4-specific viral load is greater than zero.

In a preferred embodiment, if QXR=1, almost all of the viruses in thepopulation use the R5 coreceptor; if QXR=0, almost all of the viruses inthe population use the X4 coreceptor; and if QXR<1, the viruses in thepopulation use a mixture of the R5 and X4 coreceptors.

Preferably, the patient-derived biological sample is any bodily fluid ortissue. In one embodiment, the biological sample may be a bodily fluidwhich may be selected from the group consisting of blood, plasma, andspinal fluid. In another embodiment, the biological sample may be onewhich contains viral populations that are distinct from those in thereadily obtained peripheral blood including the reservoirs of thegenital tract and lymphoid tissue.

Patient-derived biological samples may be obtained by methods known toone of skill in the art. For instance, peripheral blood of HIV-infectedindividuals can be separated into plasma and cell components by methodsknown in the art. Primary viral isolates of HIV-1 may also be obtainedby co-culture with normal donor peripheral blood mononuclear cells(PBMCs). Titration of viral isolates in PBMCs can be carried out. Thesestandard techniques are described throughout the literature; forexample, see Fang et al. (1995) Proc. Natl. Acad. Sci. USA 92:12110-4.

Preferably, the individual molecular clones each comprise a DNA sequencecorresponding to a portion of the HIV genome, the DNA sequencecomprising at least a portion of the genetic determinates of coreceptorusage.

In a preferred embodiment, the genetic determinates are derived from theenv gene. The envelope protein may comprise gp120, gp 160 or a portionthereof. Envelope sequences are predictive of coreceptor use on thebasis of the overall charge of the V3 loop and the presence of basic oracidic residues at positions 275 and 287 of the env gene (Bhattacharyaet al. (1996) AIDS Res. Hum. Retrovir. 12:83-90; Hung et al. (1999) J.Virol. 73:8216-26; and Cardozo et al (2007) AIDS Res. Hum. Retrovir. Inpress).

Cloning strategies for isolating envelope genes of interest are wellknown to one of skill in the art. See, for example, Sambrook, Fritschand Maniatis, Molecular Cloning, A Laboratory Manual, 2^(nd) Ed., ColdSpring Harbor Laboratory Press, 1989.

Preferably, the cloning methods used in the present invention willdecrease the chance of sampling error or recombination. For example,high fidelity cloning of the samples above may be achieved by routineperformance of multiple long RT-PCR reactions on limiting dilutions ofRNA, followed by multiple PCR's on cDNAs obtained from each RT reaction.In addition, performance of multiple PCR's on each cDNA preparationincreases the likelihood of amplifying a different HIV-1 RNA species.Short-term limited dilution techniques are also well known to one ofskill in the art, see for example, Connor et al. (1997). Furthermore,quantitation of HIV-1 RNA in the biological samples of the methodsdescribed herein may be carried out, for example, by using NucliSens(Organon Teknika Corp., Durham, N.C.). Quantitation methods may setouter limits. In a preferred embodiment, RNA is amplified to ≦80copies/ml.

In a preferred embodiment, the molecular clones each are derived fromRNA of the patient-derived HIV and correspond to the HIV genome or aportion thereof and which comprise the genetic determinates ofcoreceptor usage or a portion thereof. In another preferred embodiment,the molecular clones are prepared by RT-PCR of the RNA of thepatient-derived HIV and at least one set of oligonucleotide primers. Ina more preferred embodiment, at least one set of oligonucleotide primersconsists of the first set of primers in Table 3. In another morepreferred embodiment, at least one set of oligonucleotide primersincludes a second set of oligonucleotide primers, the second setconsisting of the second set of primers in Table 3. Preferably, thenumber of individual molecular clones is at least 20.

In a preferred embodiment, the heteroduplex tracking assay of the methodmay comprise the steps of: (a) amplifying the individual molecular cloneor a portion thereof by PCR to provide amplified DNA comprising thegenetic determinates of coreceptor usage or a portion thereof; (b)forming a population of heteroduplex molecules by contacting theamplified DNA with a labeled probe complementary to the amplified DNAunder conditions sufficient to form heteroduplexes; (c) separating thepopulation of heteroduplex molecules using a separation means; (d)detecting the presence or absence of heteroduplex molecules; wherein thepresence or absence of heteroduplex molecules reveals coreceptor usage.More preferably, the labeled probe may be derived from a known HIV-1CCR5 clone or from a known HIV-1 CXCR4 clone. In another preferredembodiment, the labeled probe comprises a detectable moiety, aradioisotope, biotin, a fluorescent moiety, a fluorophore, achemiluminescent moiety, or an enzymatic moiety.

The heteroduplex tracking assay of the invention can be carried outsubstantially in accordance with the guidance of Delwart et al. (J.Virol. (1994) 68:6672-6683), Delwart et al. (Science (1993)262:1257-1261), Nelson et al. (J. Virol. (1997) 71:8850-8; Delwart etal. (PCR Methods and Applications 4:S202-S216 (19950 Cold SpringsHarbor), and U.S. Pat. No. 5,851,759 (Weiner), each of which areincorporated in their entireties by reference.

The heteroduplex tracking assay can be used to analyze a portion of theHIV-1 genome encompassing determinates of coreceptor utilization tounderstand, determine, monitor, or detect coreceptor usage. Geneticdeterminates of HIV-1 coreceptor utilization can be found in theenvelope gene (env), with key determinates being found in the thirdvariable (V3) domain of the gp120 glycoprotein.

The heteroduplex tracking assay of the invention can be carried outgenerally, while not being limited thereto, according to the basic stepsof: (a) obtaining HIV viral RNA from the patient, (b) amplifying, e.g.PCR and/or reverse transcription, a portion of the viral genomecontaining genetic determinates of coreceptor usage, e.g. a genomicportion comprising the V3 domain of the gp120 envelope glycoprotein, (c)forming heteroduplexes and/or homoduplexes with labeled nucleicacid-based probes prepared from a corresponding genomic region of aknown HIV strain, e.g. the same genomic portion comprising the V3 domainof gp120, and (d) subjecting the heteroduplexes and homoduplexes to aseparation system, e.g. electrophoresis through non-denaturingpolyacrylamide gels, wherein the heteroduplexes and homoduplexes havediffering and distinguishable mobilities that results in differentmobility patterns, e.g. a electrophoretic pattern, such that thecoreceptor usage can be determined.

For example, the presence of an electrophoretic pattern characteristicof X4-heteroduplexes can indicate the presence of CXCR4-specific virusesin the HIV sample. Alternately, the presence of an electrophoreticpattern characteristic of homoduplexes and R5-heteroduplexes canindicate the presence of only CCR5-specific viruses. And, a patterncharacteristic of both homoduplexes and X4- and R5-heteroduplexes canindicate that the HIV sample contains a mixed population ofCCR5-specific and CXCR4-specific viruses. The heteroduplex trackingassay can be performed at any point during disease progression orduring, before, or after administering antiretroviral therapy. Further,the heteroduplex tracking assay can be carried out either to attainqualitative results or quantitative results.

Methods for obtaining and/or extracting HIV RNA from patient-derivedsamples are well-known. Also, the step of amplifying a portion of theviral genome containing genetic determinates of coreceptor usage a knownin the art, and include, for example reverse transcription PCR (RT-PCR).Following RT-PCT, further rounds of PCR can be used to further amplifydesired portions of the genome, especially regions containing geneticdeterminates of coreceptor usage.

It will be appreciated that the heteroduplex tracking assay is based onthe observation that when sequences were amplified by nested PCR fromperipheral blood mononuclear cells of infected individuals, related DNAproducts coamplified from divergent templates could randomly reanneal toform heteroduplexes that migrate with reduced mobility in neutralpolyacrylamide gels. Using these techniques, one can establish geneticrelationships between multiple viral DNA template molecules, such as thedifferent genetic types (i.e. different genotypes) of HIV utilizing thedifferent coreceptors. The HTA of the invention can be described asutilizing a first PCR product as a labeled probe, e.g. radioactive ornonradioactive, which is mixed with an excess (“driver”) of an unlabeledPCR product from a different source, i.e., the source for which typingor analysis of is desired, e.g. the PCR product defining the portion ofthe HIV genome with the coreceptor genetic determinates. The probesequences are then “driven” completely into heteroduplexes with thedriver, and are separated, e.g. by gel electrophoresis, on the basis ofsize. An autoradiogram or fluoroimage, for example, of the resultingpolyacrylamide gel reveals these heteroduplexes and provides a visualdisplay of the relationship between the two virus populations understudy. The fact that heteroduplexes migrate with distinct mobilitiesindicates that the strand-specific composition of mismatched andunpaired nucleotides affects their mobility.

A “heteroduplex” encompasses a doublestranded DNA molecule havingcomplementary strands at which one strand (the “target strand”, i.e. asingle strand of DNA from the PCR product of the HIV genome) containsone or more mismatched or an unpaired nucleotide base. For example, aheteroduplex can form by mixing together a labeled probe (e.g. adouble-stranded DNA PCR product of a portion of the envgene ofCCR5-specific HIV) and a PCR product of a target sequence (e.g. adouble-stranded DNA PCR product of the corresponding portion of the envgene of a CXCR4-specific HIV) such that complementary single-strandedDNA of each PCR product are combined together as a new, double-strandedmolecule. However, since the PCR product from the CXCR4-specific HIVwill contain genetic determinates characteristic of CXCR4 type viruses,its nucleotide sequence will vary at specific locations with respect tothe probe PCR product (which is derived from CCR5). These differences insequence result in a heteroduplex which has reduced mobility duringelectrophoresis with respect to homoduplexes oweing to a reduced levelof base-pairing in the molecule. In contrast, the “homoduplex” may beformed between complementary strand pairs derived from a probe PCRproduct and a target PCR product such that their nucleotide sequencesare the same.

In another embodiment, the heteroduplex tracking assay can comprise thesteps of (a) amplifying an individual molecular clone or a portionthereof by PCR to provide amplified DNA comprising the geneticdeterminates of coreceptor usage or a portion thereof; (b) forming apopulation of heteroduplex molecules by contacting the amplified DNAwith a labeled probe complementary to the amplified DNA under conditionssufficient to form heteroduplexes; (c) separating the population ofheteroduplex molecules using a separation means: and (d) detecting thepresence or absence of heteroduplex molecules; wherein the presence orabsence of heteroduplex molecules reveals coreceptor usage. In oneembodiment, the labeled probe may be derived from a known HIV-1 CCR5clone. In another embodiment, the labeled probe may be derived from aknown HIV-1 CXCR4 clone. The labeled probe can comprise a detectablemoiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, achemiluminescent moiety, or an enzymatic moiety. Appropriate labels andtheir methods of preparation are well-known.

It is furthermore envisaged that the diagnostic method may involve theuse of micro-chips comprising nucleic acid molecules encoding a envelopeprotein, or a fragment thereof, preferably a V3 region fragment,especially including genetic determinates of coreceptor usage, on genechips; or an envelope protein, or a fragment thereof, preferably a V3region fragment, on protein-chips (See U.S. Pat. Nos. 6,066,454;6,045,996; 6,043,080; 6,040,193; 6,040,138; 6,033,860; 6,033,850;6,025,601; 6,022,963; 6,013,440; 5,968,740; 5,925,525; 5,922,591;5,919,523; 5,889,165; 5,885,837; 5,874,219; 5,858,659; 5,856,174;5,856,101; 5,843,655; 5,837,832; 5,834,758; 5,831,070; 5,770,722;5,770,456; 5,753,788; 5,744,305; 5,733,729; 5,710,000; 5,631,734;5,599,695; 5,593,839; 5,578,832; and 5,556,752). Diagnostic gene chipsmay comprise a collection of polypeptides that specifically detect aenvelope protein, or fragments thereof, preferably V3 region fragments;or nucleic acid molecules that specifically detect a nucleic acidmolecule encoding a envelope protein, or fragments thereof, preferablyV3 region fragments; all of which may be used for the purposes ofdetermining coreceptor use. The envelope protein may be gp160, gp120, ora portion thereof.

It will be understood that the heteroduplex tracking assay of theinvention can be used to provide both qualitative and quantitativeinformation. First, qualitative information can be derived using the HTAof the invention by analyzing the whole HIV population derived from aninfected patient to determine whether the isolated population of HIV isCCR5-specific, CXCR5-specific, or mixture of both types. It will beappreciated that qualitative information is based on the whole orsubstantially the whole HIV population rather than individual clonestherefrom. On the contrary, quantitative information can be derivedusing the HTA of the present invention by analyzing individual HIVclones (e.g. cloned portions of the HIV genome of a plurality ofindividual HIV viruses from the isolated whole population of HIV fromthe infected patient) with respect to their coreceptor usage anddetermining a ratio of CCR5-specific to CXCR4-specific clones. In oneembodiment, the invention relates to determining the QXR ratio: thenumber of HIV clones that are identified as CCR5-specific compared tothe total number of clones analyzed. It will be appreciated that the HIVclone refers to the cloned PCR product.

FIG. 5 depicts a flow chart showing the qualitative and quantitativeaspects of the HTA of the present invention. First, HIV RNA is extractedfrom the infected patient. Next, RT-PCR is carried out to obtain HIVcDNA, from which a PCR product (i.e. PCR amplicon) containing geneticdeterminates for coreceptor usage is amplied using PCR. The PCR productis then gel purified. Presumably, the PCR product will be a mixedpopulation of molecules—those genotypic for either CCR5 or CXCR4coreceptors—whenever the isolated HIV sample contains both types ofviruses. Next, the PCR product is analyzed by the HTA of the invention,which includes generally the steps of mixing together a labeled probe(e.g. a PCR product corresponding to same region in a known CCR5 strainas the amplified target PCR amplicon to be analyzed) and the amplifiedtarget PCR amplicon to form homo- or heteroduplexes. The molecules arethen separated by gel electrophoresis, for example, on a 12%polyacrylamide gel. Electrophetic techniques are well known in the art.If the QXR<1 on the qualitative test, then a quantitative test can bedone. To perform the quantitative test the V3 portion of the HIVenvelope gene is molecularly cloned and each of 20 clones is analyzed byan individual HTA.

Exemplarly results are represented in FIG. 6. The figures shows fourpanels of schematic electropherograms. The first panel is the negativecontrol, i.e. labeled probe only. The second panel shows the result ofHTA of the V3 portion of the HIV envelope gene of a CCR5 virus. Thethird panel shows the result of HTA of a CXCR4 virus. And, the fourthpanel shows the result of HTA of a mixture of CCR5 and CXCR4 virus V3regions. Four different probes (each based on a CCR5-specific controlvirus) were used to test each HIV sample. The gels show heteroduplexband patterns for those HIV samples containing CXCR4-specific andCCR5-specific viruses.

The quantitative results of the heteroduplex tracking assay of themethod of the present invention may be assessed by statistical methodswell known to one of skill in the art. For example, QXR, the proportionof plasma HIV-1 using CCR5, may be stratified into two categories: QXR=1if all virus identified uses CCR5, and QXR<1 if X4 virus is detected.The association between virologic responses and baseline QXR may beassessed by comparing the percentages of patients with undetectableHIV-1 RNA load across the different strata by using, for example,Fischer's exact test. Further, immunologic responses across two stratamay be compared by Wilcoxon rank-sum tests. Kaplan-Meier curves and Coxproportional hazard regression models may be applied to quantify theassociation of baseline or follow-up QXR (equal 1 versus less than 1)with subsequent clinical progression, defined as a new clinicalAIDS-defining event or death.

The quantitative results of the heteroduplex tracking assay of themethod of the present invention may further be assessed by otherstatistical methods well known to one of skill in the art. For example,an additional model analyzing the relationship of X4 viral load to HIV-1disease progression may be included by stratifying the X4-specific viralload into three strata: patients without detectable X4-specific viralload (i.e., QXR=1) and patients with detectable X4 viraemia below andabove the median value of X4-specific viral loads, respectively. Tocompare the predictive capacity with the established progression markersCD4 and HIV-1 RNA load the concurrent log₂ transformed CD4 values andlog₁₀ transformed HIV-1 loads in the univariable and multivariable Coxmodels may be included. Further, the inverse probability weights may beused to adjust for sampling bias.

Preferably, STATA (Version 9.1, StataCorp, College Station, Tex.) may beused for quantitative analyses.

One of skill in the art (e.g. a physician, preferably one specializingin the treatment of infectious disease) would use appropriate judgmentand discretion in determining how often to apply the diagnostic methodsfor a patient. The frequency of application may vary, depending onvarious factors, for example, the age, sex, type of antiretroviraltherapy administered to, or stage of disease progression in, a patient.

In another preferred embodiment, the antiretroviral therapy of themethod is any suitable antiretroviral treatment regimen. Morepreferably, the antiretroviral therapy is selected from the groupconsisting of combination antiretroviral therapy (cART), proteaseinhibitors, fusion inhibitors, integrase inhibitors, coreceptor specificagents, nonnucleoside analogue reverse transcriptase inhibitors andnucleoside analogue reverse transcriptase inhibitors. Preferably, thenucleoside analogue reverse transcriptase inhibitor may be 3TC or AZT.Preferably, the nonnucleoside analogue reverse transcriptase inhibitoris nevirapine.

Antiretroviral therapy may include, but is not limited to, HAART,protease inhibitors, fusion inhibitors, integrase inhibitors,co-receptor specific agents, 3TC, AZT, nevirapine, non-nucleosideanalogue reverse transcriptase inhibitors and nucleoside analoguereverse transcriptase inhibitors. HAART can be three or moreantiretroviral drugs in combination. The term “HAART” as used hereinrefers to an combination of highly active antiretroviral agents andusually comprises three drugs.

Typical reverse transcriptase inhibitors include nucleoside analogs,such as, but not limited to, (zidovudine, (AZT, Retrovir), didanosine(ddI, Videx), stavudine, (d4T, Zerit), lamivudine, 3TC, Epivir),abacavir, (ABC, Ziagen), tenofovir, (TDF, Viread), combivir (CBV,combination of AZT and 3TC), and non-nucleoside reverse transcriptaseinhibitors, e.g., nevirapine (NVP, Viramune), delavirdine (DLV,rescriptor), efavirenz, (EFV, sustiva,). Protease inhibitors includesaquinavir, (SQV, Invirase), ritonavir (RTV, Norvir), indinavir, (IDV,Crixivan), nelfinavir, (NFV, Viracept), fosamprenivir, FPV, Lexiva),kaletra (lopinavir and ritonavir) and fortovase (saquinavir in a softgelatin form). Thus, HAART can also be “triple cocktail” therapy—a threedrug regimen to combat HIV.

The present invention further encompasses a method of monitoring theefficacy of antiretroviral therapy in a patient which may comprisedetermining the viral load of acquired immunodeficiency virus using theCXCR4 coreceptor (X4-specific viral load) in a patient-derivedbiological sample comprising the steps of: (a) screening individualmolecular clones of patient-derived acquired immunodeficiency primaryisolate with a heteroduplex tracking assay to determine the CCR5coreceptor usage and the CXCR4 coreceptor usage of each individualmolecular clone; (b) determining the proportion of HIV using the CCR5coreceptor (R5) versus the CXCR4 coreceptor (R4) wherein the proportionis expressed as a variable called the Quantity of X4 and R5 (QXR), whichrepresents the fraction of virus in a specimen using the R5 coreceptor;(c) determining coreceptor specific viral loads of the patient-derivedacquired immunodeficiency primary isolate wherein the R5-specific viralload=(VL)(QXR) and the X4-specific viral load=(VL)(1−QXR), whereinX4-specific viral load strongly predicts disease progression duringcART.

In a preferred embodiment, if QXR=1, almost all of the viruses in apopulation use the R5 coreceptor; if QXR=0, almost all of the viruses ina population use the X4 coreceptor; and if QXR<1, the viruses in apopulation use a mixture of the R5 and X4 coreceptors.

Preferably, the patient-derived biological sample is any bodily fluid ortissue. In one embodiment, the biological sample may be a bodily fluidwhich may be selected from the group consisting of blood, plasma, andspinal fluid. In another embodiment, the biological sample may be onewhich contains viral populations that are distinct from those in thereadily obtained peripheral blood including the reservoirs of thegenital tract and lymphoid tissue.

Patient-derived biological samples may be obtained by methods known toone of skill in the art. For instance, peripheral blood of HIV-infectedindividuals can be separated into plasma and cell components by methodsknown in the art. Primary viral isolates of HIV-1 may also be obtainedby co-culture with normal donor peripheral blood mononuclear cells(PBMCs). Titration of viral isolates in PBMCs can be carried out. Thesestandard techniques are described throughout the literature; forexample, see Fang et al. (1995) Proc. Natl. Acad. Sci. USA 92:12110-4.

Preferably, the individual molecular clones each comprise a DNA sequencecorresponding to a portion of the HIV genome, the DNA sequencecomprising at least a portion of the genetic determinates of coreceptorusage.

In a preferred embodiment, the genetic determinates are derived from theenv gene. The envelope protein may comprise gp120, gp 160 or a portionthereof. Envelope sequences are predictive of coreceptor use on thebasis of the overall charge of the V3 loop and the presence of basic oracidic residues at positions 275 and 287 of the env gene (Bhattacharyaet al. (1996) AIDS Res. Hum. Retrovir. 12:83-90; and Hung et al. (1999)J. Virol. 73:8216-26; and; Cardozo et al. (2007) AIDS Res. Hum.Retrovir. In press).

Cloning strategies for isolating envelope genes of interest are wellknown to one of skill in the art. See, for example, Sambrook, Fritschand Maniatis, Molecular Cloning, A Laboratory Manual, 2^(nd) Ed., ColdSpring Harbor Laboratory Press, 1989.

Preferably, the cloning methods used in the present invention willdecrease the chance of sampling error or recombination. For example,high fidelity cloning of the samples above may be achieved by routineperformance of multiple long RT-PCR reactions on limiting dilutions ofRNA, followed by multiple PCR's on cDNAs obtained from each RT reaction.In addition, performance of multiple PCR's on each cDNA preparationincreases the likelihood of amplifying a different HIV-1 RNA species.Short-term limited dilution techniques are also well known to one ofskill in the art, see for example, Connor et al. (1997). Furthermore,quantitation of HIV-1 RNA in the biological samples of the methodsdescribed herein may be carried out, for example, by using NucliSens(Organon Teknika Corp., Durham, N.C.). Quantitation methods may setouter limits. In a preferred embodiment, RNA is amplified to ≦80copies/ml.

In a preferred embodiment, the molecular clones each are derived fromRNA of the patient-derived HIV and correspond to the HIV genome or aportion thereof and which comprise the genetic determinates ofcoreceptor usage or a portion thereof. In another preferred embodiment,the molecular clones are prepared by PCR of the RNA of thepatient-derived HIV and at least one set of oligonucleotide primers. Ina more preferred embodiment, at least one set of oligonucleotide primersconsists of the first set of primers in Table 3. In another morepreferred embodiment, at least one set of oligonucleotide primersincludes a second set of oligonucleotide primers, the second setconsisting of the second set of primers in Table 3. Preferably, thenumber of individual molecular clones is at least 20.

In a preferred embodiment, the heteroduplex tracking assay of the methodmay comprise the steps of: (a) amplifying the individual molecular cloneor a portion thereof by PCR to provide amplified DNA comprising thegenetic determinates of coreceptor usage or a portion thereof; (b)forming a population of heteroduplex molecules by contacting theamplified DNA with a labeled probe complementary to the amplified DNAunder conditions sufficient to form heteroduplexes; (c) separating thepopulation of heteroduplex molecules using a separation means; (d)detecting the presence or absence of heteroduplex molecules; wherein thepresence or absence of heteroduplex molecules reveals coreceptor usage.More preferably, the labeled probe may be derived from a known HIV-1CCR5 clone or from a known HIV-1 CXCR4 clone. In another preferredembodiment, the labeled probe comprises a detectable moiety, aradioisotope, biotin, a fluorescent moiety, a fluorophore, achemiluminescent moiety, or an enzymatic moiety.

The heteroduplex tracking assay of the invention can be carried outsubstantially in accordance with the guidance of Delwart et al. (J.Virol. (1994) 68:6672-6683), Delwart et al. (Science (1993)262:1257-1261), Nelson et al. (J. Virol. (1997) 71:8850-8; Delwart etal. (PCR Methods and Applications 4:S202-S216 (19950 Cold SpringsHarbor), and U.S. Pat. No. 5,851,759 (Weiner), each of which areincorporated in their entireties by reference.

The heteroduplex tracking assay can be used to analyze a portion of theHIV-1 genome encompassing determinates of coreceptor utilization tounderstand, determine, monitor, or detect coreceptor usage. Geneticdeterminates of HIV-1 coreceptor utilization can be found in theenvelope gene (env), with key determinates being found in the thirdvariable (V3) domain of the gp120 glycoprotein.

The heteroduplex tracking assay of the invention can be carried outgenerally, while not being limited thereto, according to the basic stepsof: (a) obtaining HIV viral RNA from the patient, (b) amplifying, e.g.PCR and/or reverse transcription, a portion of the viral genomecontaining genetic determinates of coreceptor usage, e.g. a genomicportion comprising the V3 domain of the gp120 envelope glycoprotein, (c)forming heteroduplexes and/or homoduplexes with labeled nucleicacid-based probes prepared from a corresponding genomic region of aknown HIV strain, e.g. the same genomic portion comprising the V3 domainof gp120, and (d) subjecting the heteroduplexes and homoduplexes to aseparation system, e.g. electrophoresis through non-denaturingpolyacrylamide gels, wherein the heteroduplexes and homoduplexes havediffering and distinguishable mobilities that results in differentmobility patterns, e.g. a electrophoretic pattern, such that thecoreceptor usage can be determined.

For example, the presence of an electrophoretic pattern characteristicof X4-heteroduplexes can indicate the presence of CXCR4-specific virusesin the HIV sample. Alternately, the presence of an electrophoreticpattern characteristic of homoduplexes and R5-heteroduplexes canindicate the presence of only CCR5-specific viruses. And, a patterncharacteristic of both homoduplexes and X4- and R5-heteroduplexes canindicate that the HIV sample contains a mixed population ofCCR5-specific and CXCR4-specific viruses. The heteroduplex trackingassay can be performed at any point during disease progression orduring, before, or after administering antiretroviral therapy. Further,the heteroduplex tracking assay can be carried out either to attainqualitative results or quantitative results.

Methods for obtaining and/or extracting HIV RNA from patient-derivedsamples are well-known. Also, the step of amplifying a portion of theviral genome containing genetic determinates of coreceptor usage a knownin the art, and include, for example reverse transcription PCR (RT-PCR).Following RT-PCT, further rounds of PCR can be used to further amplifydesired portions of the genome, especially regions containing geneticdeterminates of coreceptor usage.

It will be appreciated that the heteroduplex tracking assay is based onthe observation that when sequences were amplified by nested PCR fromperipheral blood mononuclear cells of infected individuals, related DNAproducts coamplified from divergent templates could randomly reanneal toform heteroduplexes that migrate with reduced mobility in neutralpolyacrylamide gels. Using these techniques, one can establish geneticrelationships between multiple viral DNA template molecules, such as thedifferent genetic types (i.e. different genotypes) of HIV utilizing thedifferent coreceptors. The HTA of the invention can be described asutilizing a first PCR product as a labeled probe, e.g. radioactive ornonradioactive, which is mixed with an excess (“driver”) of an unlabeledPCR product from a different source, i.e., the source for which typingor analysis of is desired, e.g. the PCR product defining the portion ofthe HIV genome with the coreceptor genetic determinates. The probesequences are then “driven” completely into heteroduplexes with thedriver, and are separated, e.g. by gel electrophoresis, on the basis ofsize. An autoradiogram or fluoroimage, for example, of the resultingpolyacrylamide gel reveals these heteroduplexes and provides a visualdisplay of the relationship between the two virus populations understudy. The fact that heteroduplexes migrate with distinct mobilitiesindicates that the strand-specific composition of mismatched andunpaired nucleotides affects their mobility.

A “heteroduplex” encompasses a doublestranded DNA molecule havingcomplementary strands at which one strand (the “target strand”, i.e. asingle strand of DNA from the PCR product of the HIV genome) containsone or more mismatched or an unpaired nucleotide base. For example, aheteroduplex can form by mixing together a labeled probe (e.g. adouble-stranded DNA PCR product of a portion of the envgene ofCCR5-specific HIV) and a PCR product of a target sequence (e.g. adouble-stranded DNA PCR product of the corresponding portion of the envgene of a CXCR4-specific HIV) such that complementary single-strandedDNA of each PCR product are combined together as a new, double-strandedmolecule. However, since the PCR product from the CXCR4-specific HIVwill contain genetic determinates characteristic of CXCR4 type viruses,its nucleotide sequence will vary at specific locations with respect tothe probe PCR product (which is derived from CCR5). These differences insequence result in a heteroduplex which has reduced mobility duringelectrophoresis with respect to homoduplexes oweing to a reduced levelof base-pairing in the molecule. In contrast, the “homoduplex” may beformed between complementary strand pairs derived from a probe PCRproduct and a target PCR product such that their nucleotide sequencesare the same.

In another embodiment, the heteroduplex tracking assay can comprise thesteps of (a) amplifying an individual molecular clone or a portionthereof by PCR to provide amplified DNA comprising the geneticdeterminates of coreceptor usage or a portion thereof; (b) forming apopulation of heteroduplex molecules by contacting the amplified DNAwith a labeled probe complementary to the amplified DNA under conditionssufficient to form heteroduplexes; (c) separating the population ofheteroduplex molecules using a separation means: and (d) detecting thepresence or absence of heteroduplex molecules; wherein the presence orabsence of heteroduplex molecules reveals coreceptor usage. In oneembodiment, the labeled probe may be derived from a known HIV-1 CCR5clone. In another embodiment, the labeled probe may be derived from aknown HIV-1 CXCR4 clone. The labeled probe can comprise a detectablemoiety, a radioisotope, biotin, a fluorescent moiety, a fluorophore, achemiluminescent moiety, or an enzymatic moiety. Appropriate labels andtheir methods of preparation are well-known.

It is furthermore envisaged that the diagnostic method may involve theuse of micro-chips comprising nucleic acid molecules encoding a envelopeprotein, or a fragment thereof, preferably a V3 region fragment,especially including genetic determinates of coreceptor usage, on genechips; or an envelope protein, or a fragment thereof, preferably a V3region fragment, on protein-chips (See U.S. Pat. Nos. 6,066,454;6,045,996; 6,043,080; 6,040,193; 6,040,138; 6,033,860; 6,033,850;6,025,601; 6,022,963; 6,013,440; 5,968,740; 5,925,525; 5,922,591;5,919,523; 5,889,165; 5,885,837; 5,874,219; 5,858,659; 5,856,174;5,856,101; 5,843,655; 5,837,832; 5,834,758; 5,831,070; 5,770,722;5,770,456; 5,753,788; 5,744,305; 5,733,729; 5,710,000; 5,631,734;5,599,695; 5,593,839; 5,578,832; and 5,556,752). Diagnostic gene chipsmay comprise a collection of polypeptides that specifically detect aenvelope protein, or fragments thereof, preferably V3 region fragments;or nucleic acid molecules that specifically detect a nucleic acidmolecule encoding a envelope protein, or fragments thereof, preferablyV3 region fragments; all of which may be used for the purposes ofdetermining coreceptor use. The envelope protein may be gp160, gp120, ora portion thereof.

It will be understood that the heteroduplex tracking assay of theinvention can be used to provide both qualitative and quantitativeinformation. First, qualitative information can be derived using the HTAof the invention by analyzing the whole HIV population derived from aninfected patient to determine whether the isolated population of HIV isCCR5-specific, CXCR5-specific, or mixture of both types. It will beappreciated that qualitative information is based on the whole orsubstantially the whole HIV population rather than individual clonestherefrom. On the contrary, quantitative information can be derivedusing the HTA of the present invention by analyzing individual HIVclones (e.g. cloned portions of the HIV genome of a plurality ofindividual HIV viruses from the isolated whole population of HIV fromthe infected patient) with respect to their coreceptor usage anddetermining a ratio of CCR5-specific to CXCR4-specific clones. In oneembodiment, the invention relates to determining the QXR ratio: thenumber of HIV clones that are identified as CCR5-specific compared tothe total number of clones analyzed. It will be appreciated that the HIVclone refers to the cloned PCR product.

FIG. 5 depicts a flow chart showing the qualitative and quantitativeaspects of the HTA of the present invention. First, HIV RNA is extractedfrom the infected patient. Next, RT-PCR is carried out to obtain HIVcDNA, from which a PCR product (i.e. PCR amplicon) containing geneticdeterminates for coreceptor usage is amplied using PCR. The PCR productis then gel purified. Presumably, the PCR product will be a mixedpopulation of molecules—those genotypic for either CCR5 or CXCR4coreceptors—whenever the isolated HIV sample contains both types ofviruses. Next, the PCR product is analyzed by the HTA of the invention,which includes generally the steps of mixing together a labeled probe(e.g. a PCR product corresponding to same region in a known CCR5 strainas the amplified target PCR amplicon to be analyzed) and the amplifiedtarget PCR amplicon to form homo- or heteroduplexes. The molecules arethen separated by gel electrophoresis, for example, on a 12%polyacrylamide gel. Electrophetic techniques are well known in the art.If the QXR<1 on the qualitative test, then a quantitative test can bedone. To perform the quantitative test the V3 portion of the HIVenvelope gene is molecularly cloned and each of 20 clones is analyzed byan individual HTA.

Exemplarly results are represented in FIG. 6. The figures shows fourpanels of schematic electropherograms. The first panel is the negativecontrol, i.e. labeled probe only. The second panel shows the result ofHTA of the V3 region of the envelope gene of a CCR5 virus. The thirdpanel shows the result of HTA of the V3 region of the envelope gene of aCXCR4 virus. And, the fourth panel shows the result of HTA of a mixtureof CCR5 and CXCR4 virus V3 regions. Four different probes (each based ona CCR5-specific control virus) were used to test each HIV sample. Thegels show heteroduplex band patterns for those HIV samples containingCXCR4-specific and CCR5-specific viruses.

The quantitative results of the heteroduplex tracking assay of themethod of the present invention may be assessed by statistical methodswell known to one of skill in the art. For example, QXR, the proportionof plasma HIV-1 using CCR5, may be stratified into two categories: QXR=1if all virus identified uses CCR5, and QXR<1 if X4 virus is detected.The association between virologic responses and baseline QXR may beassessed by comparing the percentages of patients with undetectableHIV-1 RNA load across the different strata by using, for example,Fischer's exact test. Further, immunologic responses across two stratamay be compared by Wilcoxon rank-sum tests. Kaplan-Meier curves and Coxproportional hazard regression models may be applied to quantify theassociation of baseline or follow-up QXR (equal 1 versus less than 1)with subsequent clinical progression, defined as a new clinicalAIDS-defining event or death.

The quantitative results of the heteroduplex tracking assay of themethod of the present invention may further be assessed by otherstatistical methods well known to one of skill in the art. For example,an additional model analyzing the relationship of X4 viral load to HIV-1disease progression may be included by stratifying the X4-specific viralload into three strata: patients without detectable X4-specific viralload (i.e., QXR=1) and patients with detectable X4 viraemia below andabove the median value of X4-specific viral loads, respectively. Tocompare the predictive capacity with the established progression markersCD4 and HIV-1 RNA load the concurrent log₂ transformed CD4 values andlog₁₀ transformed HIV-1 loads in the univariable and multivariable Coxmodels may be included. Further, the inverse probability weights may beused to adjust for sampling bias.

Preferably, STATA (Version 9.1, StataCorp, College Station, Tex.) may beused for quantitative analyses.

One of skill in the art (e.g. a physician, preferably one specializingin the treatment of infectious disease) would use appropriate judgmentand discretion in determining how often to apply the diagnostic methodsfor a patient. The frequency of application may vary, depending onvarious factors, for example, the age, sex, type of antiretroviraltherapy administered to, or stage of disease progression in, a patient.

In another preferred embodiment, the antiretroviral therapy of themethod is any suitable antiretroviral treatment regimen. Morepreferably, the antiretroviral therapy is selected from the groupconsisting of combination antiretroviral therapy (cART), proteaseinhibitors, fusion inhibitors, integrase inhibitors, coreceptor specificagents, nonnucleoside analogue reverse transcriptase inhibitors andnucleoside analogue reverse transcriptase inhibitors. Preferably, thenucleoside analogue reverse transcriptase inhibitor may be 3TC or AZT.Preferably, the nonnucleoside analogue reverse transcriptase inhibitoris nevirapine.

Antiretroviral therapy may include, but is not limited to, HAART,protease inhibitors, fusion inhibitors, integrase inhibitors,co-receptor specific agents, 3TC, AZT, FTC, efavirenz, nevirapine,non-nucleoside analogue reverse transcriptase inhibitors and nucleosideanalogue reverse transcriptase inhibitors. HAART can be three or moreantiretroviral drugs in combination. The term “HAART” as used hereinrefers to a combination of highly active antiretroviral agents andusually comprises three drugs

Typical reverse transcriptase inhibitors include nucleoside analogs,such as, but not limited to, zidovudine, (AZT, Retrovir), didanosine(ddI, Videx), stavudine, (d4T, Zerit), lamivudine, 3TC, Epivir),abacavir, (ABC, Ziagen), tenofovir, (TDF, Viread), combivir (CBV,combination of AZT and 3TC), and non-nucleoside reverse transcriptaseinhibitors, e.g., nevirapine (NVP, Viramune), delavirdine (DLV,rescriptor), efavirenz, (EFV, sustiva,). Protease inhibitors includesaquinavir, (SQV, Invirase), ritonavir (RTV, Norvir), indinavir, (IDV,Crixivan), nelfinavir, (NFV, Viracept), fosamprenivir, FPV, Lexiva),kaletra (lopinavir and ritonavir) and fortovase (saquinavir in a softgelatin form). Thus, HAART can also be “triple cocktail” therapy—a threedrug regimen to combat HIV.

The present invention further encompasses a diagnostic compositioncomprised of the methods of the present invention in the form of a kit.The diagnostic composition may comprise the components as defined hereinabove wherein said components are bound to/attached to and/or linked toa solid support. It is furthermore envisaged, that the diagnosticcomposition may comprise nucleic acid sequences encoding an envelopeprotein, or a fragment thereof, preferably a V3 region fragment; orindicator cell lines of this invention; all of which may be contained onmicro-chips identifiable with a suitable means for detection.

Solid supports are well known in the art and comprise, inter alia,commercially available column materials, polystyrene beads, latex beads,magnetic beads, colloid metal particles, glass and/or silicon chips andsurfaces, nitrocellulose strips, membranes, sheets, duracytes, wells andwalls of reaction trays, plastic tubes etc. Suitable methods forfixing/immobilizing cells, nucleic acid sequences, or polypeptides ofthe invention are well known and include, but are not limited to ionic,hydrophobic, covalent interactions and the like.

The diagnostic composition of the present invention may be used as akit, inter alia, for carrying out the methods of the present invention,for example diagnostic kits or research tools. Additionally, the kit ofthe invention may contain suitable means for any other scientific,medical and/or diagnostic purposes.

Diagnostic compositions and kits of the present invention may bemanufactured by standard procedures that are well known to one of skillin the art. Kits may advantageously include instructions for use and/oradmixture of ingredients.

One of skill in the art appreciates that the diagnostic compositions andkits of the present invention are not limited to use with HIV, but maybe used, based on the teachings herein and knowledge of one of skill inthe art, to identify and quantitate analogous coreceptors of otherlentiviruses, such as SIV and FIV. (See, for example, U.S. Pat. Nos.5,863,542 and 5,766,598).

The present invention is additionally described by way of the followingillustrative, non-limiting Examples, that provide a better understandingof the present invention and of its many advantages.

EXAMPLES

The Examples show that HAART not only reduces the quantity of virus butalso affects HIV-1 coreceptor use. Briefly, methods were devised forquantifying the proportion of viruses in patient-derived virus that usedeach coreceptor and monitoring the effect of combination antiretroviraltherapy, particularly HAART, on coreceptor use. The Examples furthershow that QXR and X4-specific viral load are predictors of diseaseprogression and clinical outcome.

Example 1 Study Population

Coreceptor use was examined in twenty-two women who participated in twoprospective studies of HIV-1 infection. Nineteen were enrolled in theBronx-Manhattan site of Women's Interagency HIV Study (WIHS), a NationalInstitutes of Health (NIH) multicenter study of the natural history ofHIV-1 infection in women. Three took part in a study of HIV-1pathogenesis performed at the Wadsworth Center of the New York StateDepartment of Health in Albany, N.Y. Both studies included individualswith a broad spectrum of HIV-1 disease. The institutional review boardsat each clinical site and the New York State Department of Healthapproved the investigation. Each woman provided informed consent atenrollment.

To examine the effect of combination antiretroviral therapy on HIV-1coreceptor use, women infected with CXCR4 strains were sought. Afterscreening twenty-two women, most with advanced HIV-1 disease, fifteenparticipants meeting the following criteria were studied: 1) viralisolates displayed CXCR4 strains while untreated or taking nucleosideanalogues alone; and 2) antiretroviral therapy, when initiated, wasdocumented by the WIHS database, Wadsworth study questionnaires, andrecords of treating physicians.

Sample Collection, Preparation, and Analysis

Once the study population was selected, blood was drawn and separatedinto plasma and cell components (Anastos et al. (2000) J. AIDS Hum.Retro. (in press); Fang et al. (1995)). HIV-1 RNA in plasma wasquantitated by using NucliSens (Organon Teknika Corp., Durham, N.C.),with a lower limit of quantitation of ˜80 copies/ml. The CCR5 genotypeof each patient was determined as described (Samson et al. (1996)).

Derivation of Primary Viral Isolates and Biological Clones

Primary isolates of HIV-1 were obtained by co-culture with normal donorPBMCs. Fang et al. (1995). Viral isolates were titrated in PBMCs (Fanget al. (1995)). Biological clones were derived from primary isolates byshort-term limiting dilution cloning (Connor et al. (1997)).

Patient Population and Response to Therapy

Initially, most of the fifteen women displayed high plasma HIV-1 RNAlevels and CD4+ cell depletion (means of 5.22 log₁₀ copies/ml and 147cells/mm³, respectively). At that time, eight women were receivingantiretroviral therapy, primarily zidovudine monotherapy. While understudy, however, 12 initiated new combination regimens; 9 received HAART(Group I) and 3 received two or more nucleoside analogues (Group II).Three individuals, by contrast, did not initiate new therapy during thestudy (Group III) (Table 1). In Table 1, “Before therapy” refers to dataobtained at the visit immediately preceding initiation of new two orthree drug antiretroviral therapy in Groups I & II. For Group III, datafrom the first time point are shown (a). “Follow-up” refers to dataobtained at the first time point following the initiation of theanti-HIV therapy listed for Groups I & II. For Group III, data from thefinal time point are displayed (b). Comparisons of QXR before and afterinitiation of new, combination antiretroviral therapy were statisticallysignificant for Group I, HAART recipients (c), (P=0.023) and Groups I &II combined, consisting of all treated patients (P=0.003).

TABLE 1 Patient Characteristics Before and After Antiretroviral TherapyStatus Before Combination Therapy^(a) Follow-up Status^(b) HIV-1 CD4+QXR, HIV-1 CD4+ QXR, RNA, log count, proportion RNA, log count,proportion copies per cells per Anti-HIV of HIV-1 copies per cells perAnti-HIV of HIV-1 Pt. ml mm3 Therapy Using R5 ml mm3 Therapy Using R5Group I: HAART Recipients 1 5.30 188 AZT 0.36 5.08 578 3TC, d4T, 1.00Nel 2 5.69 3 None 0.00 3.41 90 3TC, d4T, 1.00 Nel 3 5.75 291 None 0.344.54 370 AZT, 0.45 3TC. Saq 4 5.28 9 d4T 0.36 3.08 15 3TC, d4T, 0.36 Rit5 6.08 41 None 0.36 4.96 11 3TC, d4T, 0.90 Saq 6 5.11 19 None 0.45 3.7024 3TC, d4T, 1.00 Ind 7 4.94 42 AZT 0.36 5.61 10 3TC, d4T, 0.36 Ind 85.65 0 AZT, ddl 0.44 5.29 23 3TC, d4T, 1.00 Ind 9 5.58 259 AZT 0.90 4.86282 3TC, d4T, 1.00 Ind Group II: Recipients of CombinationAntiretroviral Therapy 10  5.04 307 AZT 0.00 4.58 378 3TC, ddl 1.00 11 5.10 222 AZT, ddl 0.00 4.94 213 AZT, 0.36 3TC, d4T 12  5.04 251 None0.36 4.23 345 AZT, 3TC 1.00 Group III: Recipients of No Therapy or AZTMonotherapy 13  4.32 191 None 0.45 4.13 184 None 0.36 14  4.28 670 None0.52 3.83 429 None 0.36 15  5.23 43 AZT 0.00 5.36 NA None 0.00 MeanValues for Treatment Groups Group I 5.49 94 0.40 4.50 155 0.74_(c) GroupII 5.06 260 0.12 4.58 312 0.79 Group I & 5.38 136 0.33 4.52 194 0.75_(c)II, Combined Group III 4.61 301 0.32 4.44 307 0.24

For those initiating new therapy, HIV-1 RNA levels dropped by an averageof 0.86 log₁₀ copies/ml and CD4+ counts increased by an average of 58cells/ml by the first study visit after starting the new regimens. Theviral levels rebounded by 0.69 log₁₀ copies/ml, however, by the end ofthe 28.5 month mean follow-up period for treated patients, at which time11 of the 12 women continued to take antiretroviral therapy (6 HAART, 5two drug regimens).

Assay for Coreceptor Use

Changes in coreceptor use of primary HIV-1 isolates and biologicalclones obtained from participants in the study over time were followedby using a HOS-CD4+ cell system. The parental HOS-CD4+ line is a humanosteogenic sarcoma cell line stably expressing high levels of CD4.HOS-CD4+ cells transfected with genes encoding either CCR5 or CXCR4 inaddition to CD4 (cell lines HOS-CD4.CCR5 and HOS-CD4.CXCR4 respectively)served as indicator lines for coreceptor use. Deng et al. (1996). Todetermine coreceptor use, HOS-CD4.CCR5 and HOS-CD4.CXCR4 cells wereseeded onto 12-well plates and, after 24 hours, inoculated with astandard quantity of titered virus; 10² TCID₅₀ of first passage primaryviral isolates or biological clones were assayed in duplicate. HIV JR-FLand LAV/HTLV-IIIB inoculated in parallel as CCR5- and CXCR4-specificpositive control viruses, respectively, and uninoculated cells were usedas negative controls. To eliminate any artifacts resulting frominfection via low levels of endogenous coreceptor expression, parentalHOS-CD4+ cells were also inoculated with duplicate primary and controlisolates.

Supernatants were harvested at day 10 after infection and analyzed forHIV-1 p24 antigen using a commercially available ELISA assay (NEN LifeScience Products, Boston). ELISA values were standardized so that 0pg/ml was set at the level equal to three times the mean value of thenegative controls. A culture was considered positive if the p24 antigenlevel was equal to or greater than 25 pg/ml. Experimental results werediscarded if: 1) any parental HOS-CD4+ culture tested positive; or 2)any JR-FL or LAV/HTLV-IIIB positive control culture tested negative. Ifthe variance in p24 antigen level between duplicate cultures was greaterthan 25%, the coreceptor use assay for that particular viral isolate wasrepeated. Results of the coreceptor use assay were then categorized in asemiquantitative manner according to p24 antigen level as follows:negative (p24<25 pg/ml), +/−(25-50 pg/ml), 1+(50-250 pg/ml), 2+(250-500pg/ml), and 3+(≧500 pg/ml).

Phenotypic Characterization

The presence of syncytium-inducing (SI) variants of HIV-1 in patientprimary viral isolates was determined by infection of MT-2 cell culturesas previously described (Koot et al. (1993)). A pooled stock of HIVLAV/HTLVIII was used as a positive control.

Example 2 Antiretroviral Therapy Preferentially Suppresses CXCR4 Strains

Fourteen women initially displayed viral populations composed of bothCCR5 and CXCR4 viruses (FIG. 1) and one displayed virus that exclusivelyused CXCR4. CXCR4 viruses persisted at subsequent time points inpatients who did not initiate new combination therapy, a findingexemplified in FIG. 1 by Patient 13, who remained untreated throughoutthe study, and Patients 1, 2, and 8, whose virus was sampled on multipleoccasions before new therapy commenced. Viruses using CXCR4 appeared tobe preferentially suppressed, however, when new regimens were initiated.Not only were CXCR4 strains eliminated by the first time point afterstarting new therapy in half of the treated women (FIG. 1, Patients 1,2, 6, 8, and 10), but the proportion of these viruses seemed to bediminished in most of the others. In addition, patients who experienceda rebound in HIV-1 RNA levels and CXCR4 strains while on therapy oftenachieved suppression of CXCR4 strains a second time when theantiretroviral regimen was changed (FIG. 1, Patients 2 and 8).

Coreceptor Use by Biologically Cloned Viruses

Delineation of the proportion of individual viruses using eachcoreceptor was prompted by two aspects of the pattern of HIV-1coreceptor use in these individuals. First, analyses of primary viralisolates by the HOS-CD4+ system indicated coreceptor use by both CCR5and CXCR4 viruses at many time points (FIG. 1). Because primary isolatescomprise a molecular mixture of viral quasispecies, inventors wished todetermine whether use of both coreceptors was due to dual tropic virusesor a mixture of individual viruses with CCR5 and CXCR4 tropisms. Inaddition, to compare coreceptor use rigorously over time, it isdesirable to quantitate the proportion of virus using each coreceptor.For these reasons, biologic clones, which were derived from thepatients' primary isolates by performing limiting dilution cultures,were isolated. Coreceptor use was then determined for 25 clones fromeach isolate by employing the HOS-CD4+ cell system. Biologic clones fromthese patients used either CCR5 or CXCR4; no dual tropic viruses weredetected among the 525 clones by using our assay system. In addition,the distribution of coreceptor use by the clones generally confirmed thesemiquantitative results obtained for primary isolates; proportions ofHIV-1 using each coreceptor appeared roughly similar whether the clonedvirus or primary isolates were examined (Table 2A, HIV-1 coreceptor usein primary viral isolates and biologic clones).

TABLE 2A Co-Receptor Distribution Use of Co- Months of Primary ReceptorUse by After Viral Isolates Biologic Clones Pt Baseline Treatment CCR5CXCR4 CCR5 CXCR4 2 16 AZT, 3TC +++ +++ 8 17 18 HAART + − 25 0 26 HAART ++++ 4 21 5 0 None ++ +++ 11 14 6 HAART +++ + 21 4 9 d4T, Ind +++ +++ 1015 16 HAART +++ − 25 0 14 0 None +++ ++ 13 12 7 None +++ +++ 9 16

In Table 2A coreceptor use was determined for the primary viral isolateobtained at each time point and for 25 biologic clones derived from eachisolate.

Studies of biologic clones obtained at serial time points also confirmedthat the predominant viral population shifted from CXCR4 to the lesspathogenic CCR5 after initiating a change in the regimen of combinationantiretroviral therapy (Table 2A). For example, analyses of virusobtained from Patient 2 sixteen months after baseline and eight monthsafter initiation of double therapy showed only eight clones that usedCCR5 as compared to seventeen that used CXCR4. After a switch to a HAARTregime that included two new drugs, however, the viral population inthis patient shifted and all 25 biologic clones used CCR5. A similarpattern was exhibited by biologic clones from Patient 5, whose virusshifted dramatically to CCR5 on the two occasions that HAART wasinitiated. Patient 14, by contrast, remained untreated and her viralpopulation evolved to comprise a larger proportion of clones using CXCR4over time.

The MT2 assay to detect SI viruses in culture was also performed onprimary isolates derived at each time point. These results confirmed thepattern of HIV-1 coreceptor use described here. Thirteen of the fifteenpatients were infected initially with SI virus. In all eleven of thosewho displayed SI virus and received new combination therapy, thephenotype changed at least transiently to non-syncytia inducing (NSI)after treatment (data not shown).

Sequence Analyses of the HIV-1 V3 Loop

HIV-1 virions were isolated from plasma samples as described (Fang etal. (1996) J. AIDS Hum. Retro. 12:352-7). Reverse transcriptasepolymerase chain reaction amplification produced a 920-bp ampliconspanning the V3 region of the env gene. Reaction conditions werecontrolled rigorously to minimize recombination and other artifacts(Fang et al. (1996)). Amplified products were cloned into a TOPO™ TAvector (Invitrogen, Carlsbad, Calif.), verified by restrictiondigestion, and sequenced. Alignment of the sequences was initially doneusing the PILEUP program in the GCG Suite (Genetics Computer Group,Madison, Wis.), then checked manually. Envelope sequences were used topredict coreceptor use on the basis of the overall charge of the V3 loopand the presence of basic or acidic residues at positions 275 and 287 ofthe env gene (Bhattacharyya et al. (1996); and Hung et al. (1999)).

Coreceptor Use Determined by Sequence Analysis of HIV-1 RNA MolecularClones

These sequences predicted a pattern of coreceptor use that essentiallyparalleled the one obtained by using viral culture (Table 2B, Coreceptoruse determined by cocultivation of PBMCs vs. sequence analysis of plasmaHIV-1 RNA). Table 2B shows a comparison of coreceptor use over timedetermined by two methods in representative study patients. At each timepoint, coreceptor use was assayed by co-cultivating PBMCs anddetermining the V3 loop sequence of virion-derived HIV-1 RNA.

The sequence data underscored the change in coreceptor use seen afterinitiation of treatment. These experiments suggest that study ofcultivated virus reflects the coreceptor use of currently replicatingvirus and is likely to reveal the shifts in viral populations that occuras a result of recent antiretroviral therapy.

TABLE 2B Distribution of Co-Receptor Co-Receptor Use Use Predicted byMonths by V3 Loop Sequences After Cocultivated Total Base- Virus # of Ptline Treatment CCR5 CXCR4 CCR5 CXCR4 Clones 1 6 AZT +++ +++ 9 4 13 33HAART +++ − 13 0 13 36 HAART +++ + 8 2 10 2 16 AZT, 3TC +++ +++ 1 13 1422 HAART + ++ 0 13 13 26 HAART + +++ 3 8 11 5 0 None ++ +++ 2 10 12 6HAART +++ + 8 3 11 9 d4T, Ind +++ +++ 2 10 12 16 HAART +++ − 12 0 12 140 None +++ ++ 5 6 11

Statistical Methods

The Wilcoxon Rank Sum Test was used to make comparisons between themagnitude of log viral level, CD4+ counts, and QXR values. Data forfactors relating to changes in QXR values were analyzed by multivariatePoisson regression. Variables included log HIV-1 RNA levels, changes inviral levels, CD4+ cell counts, changes in CD4+ cell counts, andindicator variables for levels of antiretroviral therapy.

To quantitate HIV-1 coreceptor use, inventors constructed a variable, λ,as the proportion of strains using CCR5. This variable has since beenrenamed QXR. QXR=1 represents an isolate in which all strains prefer theCCR5 coreceptor but QXR=0 indicates that all prefer CXCR4. QXR valueswere assessed by utilizing qualitative assay data derived from primaryisolates, biologic clones, and sequences of the V3 portion of the envgene. In determination of the coreceptor use of 525 biologic clones,none was dual tropic, suggesting that true dual tropic viruses are rarewhen using our assay method. It was therefore assumed for thiscalculation that the probability of a single virion possessing thephenotypic attributes of both coreceptors is small. Thus, for the vastmajority of virions, each virion uses either CCR5 or CXCR4. Thisrelationship can be stated as a mixture

D=QXR(CCR5)+(1-QXR)(CXCR4); 0≦QXR≦1, where D is the distribution ofviral phenotypes. By design, it is a binomial population.

QXR values were constructed by relating data derived from the samepatient sample by using three different analyses: biologic cloning, V3sequencing of patient-derived molecular clones, and qualitative assaysof primary isolates. To construct QXR values, inventors first calculatedthe proportion of biologic and, if available, molecular clones usingCCR5 at each time point, then linked the proportion to the qualitativecoreceptor use score (− to 3+) of primary isolates obtainedsimultaneously. Data that were not available were interpolated. The datawere transformed to approximate a Poisson distribution. Poissonregression analysis was then performed to determine the factorsassociated with changes in QXR values.

Quantitation of Coreceptor Use by CCR5 and CXCR4

The large number of biologic and molecular clones permitted derivationof a system to quantitate the proportion of virus in a clinical specimenthat uses each coreceptor. In this system, QXR is a continuous,nonlinear variable between one and zero derived from the resultspresented here showing coreceptor use by biologically and molecularlycloned virus; it describes the mixed proportion of viruses using CCR5and CXCR4. A QXR value near one describes a population of viruses thatalmost all use CCR5; a value near zero describes a population thatalmost all use CXCR4. By applying this method, it was determined theproportion of virus using each coreceptor for each patient over time.

To quantitate the effect of combination therapy on HIV-1 coreceptor use,inventors compared the QXR values of virus obtained at the visits beforeand immediately after initiating new combination therapy. Thiscomparison demonstrated a clear, statistically significant shift of thepredominant viral population from CXCR4 to CCR5 (Table 1). The mean QXRvalues for virus from all twelve patients starting combination therapy(Groups I & II) changed from 0.33 to 0.75 (P=0.003 by using the binomialproportion comparison test). For the subset of nine who initiated HAART(Group I), the shift in QXR extended from 0.40 to 0.74 (P=0.023). Inaddition, inventors assessed separately the effect of initiatingtreatment with two or more nucleoside analogues and no proteaseinhibitor on coreceptor use. Five of the patients who ultimatelyreceived HAART had received regimens consisting of two nucleosideanalogues previously. The QXR values of virus obtained before or afterinitiation of two or more nucleoside analogues in a group of eightpatients (Group II and Patients 1, 2, 6, 7, and 9) were compared; inthis group the QXR values changed from 0.30 to 0.84 (P=0.008). Bycontrast, in the Group III patients, who did not initiate combinationtherapy, the mean QXR value decreased from 0.32 to 0.24 during thecourse of this study. These numerical comparisons of coreceptor usedemonstrated a shift in the predominant viral population from CXCR4 toCCR5 following initiation of a variety of combination antiretroviralregimens.

Long-Term Analysis of Antiretroviral Therapy, Viral Level, and CD4+ CellCount Effects on Coreceptor Use

The period of follow-up for treated women in this study averaged 28.5months, during which their coreceptor use, plasma HIV-1 RNA levels, andCD4+ cell count varied, sometimes in concert (FIG. 1). The mulitvariateregression indicated that antiretroviral therapy with two or more drugswas by far the most significant factor in determining QXR, the numericalexpression of the proportion of viruses using CCR5 (P=0.01). Althoughchanges in viral level and CD4+ cell count had a significant effect onQXR in univariate analysis, they lost all significance when consideredin a multivariate regression analysis with antiretroviral therapy. Thestrength of the relationship between initiation of therapy and shift inHIV-1 coreceptor use is reflected in the course of treated individualslike Patient 8, who maintained high plasma HIV-1 RNA levels duringtreatment but demonstrated a substantial, long-term shift in viralpopulation toward CCR5 (FIG. 1).

Example 3 Dynamics of HIV-1 Coreceptor Utilization Switch

The dynamics of the shift in coreceptor utilization immediatelyfollowing initiation of HAART have been characterized. Coreceptorutilization immediately following the initiation of HAART was determinedby studying virus derived from the patient's PBMC's. Results show thefollowing: 1) this patient was unusual in that her initial viralpopulation was composed of X4 viruses only, 2) by the third day afterthe initiation of HAART, the viral population had switched to equalproportions of X4 and R5 using strains, and 3) by day 11, the populationhad entirely switched to R5 using virus (FIG. 2).

Comparison of coreceptor usage in this patient was also performed usinga recombinant assay that does not require culturable primary isolates.The results of the recombinant assay were identical to the resultsobtained using virus derived from the patient's PBMC's. These datadocument a rapid, complete switch in coreceptor utilization by virus inperipheral blood that occurred less than two weeks after initiatingHAART. To understand the complexities of HIV-1 pathogenesis, it isnecessary to consider the heterogeneity of viral populations and viralreservoirs. This approach will provide insight into the dynamics ofsuppressing different populations of virus.

Example 4 Rapid Cell Fusion Assay for Coreceptor Utilization

Viral coreceptor usage was separately evaluated through the use of aRapid Cell Fusion Assay. This assay enables determination of coreceptorusage from cloned HIV env gene sequences obtained directly from patientsamples (e.g. blood, mucosal tissue). This method allows for greaterefficiency in determination of viral coreceptor usage, by circumventingthe need for cultivation of primary isolates. The Rapid Cell FusionAssay can advantageously produce a result within one week afterobtaining a patient sample. In addition, the Rapid Cell Fusion Assayallows study of patient-derived virus obtained from sites other than theperipheral blood, particularly those sites from which cultured viruscannot be obtained. For example, while circulating macrophages and CD4⁺T cells are the dominant reservoir of HIV-1, viral populations distinctfrom those in the peripheral blood exist in many reservoirs, includingthe genital tract. It is important to study these different reservoirsas HIV-1 viral populations in infected individuals demonstrate markedheterogeneity, with virus varying in the same compartment over time andin different compartments contemporaneously (Myers et al. (1995);Meyerhans et al. (1989); Vemazza et al. (1994); Cheng-Mayer et al.(1989); Koyanagi et al. (1987); ); Kemal et al. (2003)). Even inpatients receiving combination anti-HIV-1 therapy, studies of lymphoidtissue reservoirs showed persistent viral replication in lymph nodes,with viral load in tissue exceeding that in plasma by orders ofmagnitude in most cases (Wong et al. (1997); Cavert et al. (1997); Haaseet al. (1996)).

Steps of the Rapid Cell Fusion Assay

The HL3T1 cell line was derived by stable transfection of parental HeLacells with a chloramphenicol acetyltransferse (CAT) reporter constructcontaining a CAT gene is linked to an HIV-1 LTR promoter. The HL3T1cells produce CAT protein only upon introduction of an active HIV-1 Tatprotein. HL3T1 cells were transfected with a cloned env gene derivedfrom a patient of interest. The cloned env gene product is expressed onthe surface of the HL3T1 cells.

Indicator cell lines GHOST.CCR5 and GHOST.CXCR4 (respectivelyhereinafter “R5-tat” and “X4-tat”) cells were transfected withpSV2tat72, a construct expressing high levels of HIV-1 Tat under thecontrol of the SV40 early promoter.

HL3T1 cells containing a cloned patient env gene were fused to R5-tatand X4-tat cells. Cell surface envelope protein variants willselectively interact with either CCR5 or CXCR4. Fusion only occurs whenan HL3T1 envelope protein interacts with an indicator cell expressing acompatible coreceptor. Therefore, HL3T1 cells will fuse with eitherR5-tat and X4-tat, depending on the patient's env gene specificity. Toinitiate fusion, transfected HL3T1 and R5-tat or X4-tat cells were mixedin 6-well plates at 37° C. and allowed to fuse for 48 hours. Toquantitate fusion, the cells were lysed with 0.5% NP-40. Fusion of HL3T1cells to R5-tat or X4-tat activated CAT gene expression. Aliquots of thecell lysates were monitored for CAT production using a commerciallyavailable kit (CAT-ELISA, Boehringer Mannheim).

Twenty-five clones from each sample were analyzed to ensure that thefusion assay reflected the heterogeneous nature of HIV-1 populations.Sample results of the Rapid Cell Fusion Assay for Coreceptor Utilizationare presented below. For all env clones assayed in this manner, sequenceanalysis has revealed a 97% correlation between coreceptor usage andpredicted env genotype.

CLONE V3 LOOP SEQUENCE CORECEPTOR AF2P12-1CIRPNNNTRTSIRIGPGQAFYATGNIIGDIRQAYC CCR5 (SEQ ID No.1) AF2P12-2CIRPNNNTRTSIRIGPGQAFYATGNIIGGIRQAYC CCR5 (SEQ ID NO.35) AF2P12-3CIRPNNNTRTSIRIGPGQAFYATGNIIGDIRQAYC CCR5 (SEQ ID NO.1) AF2P12-4CIRPNNNTRTSIRIGPGQAFYATGNIIGDIRQAYC CCR5 (SEQ ID NO.1) AF2P12-6CIRPNNNTRTSIRIGPGQAFYATGNIIGDIRQAYC CCR5 (SEQ ID NO.1) AF2P12-8CIRPNNNTRTSIRIGPGQAFYATGNIIGDIRQAYC CCR5 (SEQ ID NO.1) AF2P12-9CIRPNNNTRTSIRIGPGQAFYATGNIIGDIRQAYC CCR5 (SEQ ID NO.1) AF2P12-10CIRPNNNTRTSIRIGPRQAFYATGNIIGDIRQAYC CXCR4 (SEQ ID No.2) AF2P12-11CIRPNNNTRTSIRIGPGQAFYATGNIVGDIRQAYC CCR5 (SEQ ID NO.3) AF2P12-12CIRPNNNTRTSIRIGPGQAFYATGNIIGDIRQAYC CCR5 (SEQ ID NO.1) AF3P-2........RKSVHIGPGQAFYATGDIIGNIRKAHC negative (SEQ ID NO.4) AF3P-4CTRPNNNTRKSVHIGPGQAFYATGDIIGNIRKAHC CCR5 (SEQ ID NO.5) AF3P-5CTRPNNNTRKSVHIGPGQAFYATGDIIGNIRKANC CCR5 (SEQ ID NO.5) AF3P-6CTRPNNNTRKSVHIGPGQAFYATGDIIGNIRQAHC CCR5 (SEQ ID NO.6) AF3P-7CTRPNNNTRKSVHIGPGQAFYATGDIIGNIRKAHC CCR5 (SEQ ID NO.5) AF3P-8CTRPNNNTRKSVHIGPGQAFYATGDIIGNIRKAHC CCR5 (SEQ ID NO.5) AF3P-9CTRPNNNTRKSVHIGLGQAFYATGDIIGNIRKAHC CCR5 (SEQ ID NO.36) AF3P-10CTRPNNNTRKSVHIGPGQAFYATGDIIGNIRKAHC CCR5 (SEQ ID NO.5) AF3P-11CTRPNNNTRKSVHIGPGQAFYATGDILGNIRQAHC CCR5 (SEQ ID NO.37) AF3P-12CTRPNNNTRKSVHIGPGQAFYATGDIIGNMRKAHC CCR5 (SEQ ID NO.7) AF5P-5CTRPNNNTRKSVHIGPGQAFYATGDIIGDIRQAYC CCR5 (SEQ ID NO.38) AF5P-6CTRPNNNTKKSVHIGPGQAFYATGDIIGDIRQAYC CCR5 (SEQ ID NO.39) AF5P-8CTRPNNNTRKSVHIGPGQAFYATGDIIGDIRQAYC CCR5 (SEQ ID NO.38) AF6P-1CTRPINNRRKSIHMGPGQAFYGT.DDIIGDIRKARC CCR5 (SEQ ID NO.8) AFGP-3CTRPINNRRKSIHMGPGQAFYGT.DDIIGDIRKARC CCR5 (SEQ ID NO.8) AF6P-7CTRPSNNRRKSIHKGDQDKHSMEHDDVIGDIRKARC negative (SEQ ID NO.9) AF6P-9CTRPINNRRKSIHMGPGQAFYGT.DDIIGDIRKARC CCR5 (SEQ ID NO.8) AFGP-10CTRPINNRRKSIHIGPGQAFYGT.DDIIGDIRQAHC CCR5 (SEQ ID NO.32) AFGP-11CTRPSNNRRKSIHMGPGQAFYGT.DDIIGGIRKARC CCR5 (SEQ ID NO.33) AF6P-12CTRPSNNRRKSIHMGPGQAFYGT.DDIIGDIRKARC CCR5 (SEQ ID NO.34) AF7P-9CIRPNNNTRQSVHIGPGQALYTTEIIGDIRKAHC CCR5 (SEQ ID NO.11) AF7P-12CIRPNNNTRQSVHIGPGQALYTTEIIGDIRKAHC CCR5 (SEQ ID NO.11) AF9P2-3CTRPNNNTITSIRIGPGQAFYATGSIIGNIRQAHC CCR5 (SEQ ID NO.12) AF9P2-4CTRPNNNTITSIRIGPGQAFYATGSIIGNTRQAHC CCR5 (SEQ ID NO.13) AF9P2-7CTRPNNNTITSIRIGPGQAFYATGSIIGNIRQAHC CCR5 (SEQ ID NO.12) AF9P2-9CTRPNNNTITSIRIGPGQAFYATGSIIGNIRQAHC CCR5 (SEQ ID No.12) AF9P2-10CTRPNNNTITSIRIGPGQAFYATGSIIGNIRQAHC CCR5 (SEQ ID NO.12) AF9P2-11CTRPNNNTITSIRIGPGQAFYATGSIIGNIRQAHC CCR5 (SEQ ID NO.12) AF9P2-12CTRPNNNTITSIRIGPGQAFYATGSIIGNIRQAHC CCR5 (SEQ ID NO.12) AF10P97-2CTRPNDNIRKSVHIGPGQAFYATGDIIGDIRRAHC CCR5 (SEQ ID NO.14) AF10P97-4CTRPNDNIRKRVHIGPGQAFYATGDVIGDIRRAHC CXCR4 (SEQ ID NO.40) AF10P97-6CTRPNDNIRKSVHIGPGQAFYATGDIIGDIRRAHC CCR5 (SEQ ID NO.14) AF10P97-11CTRPNDNIRKSVHIGPGQAFYATGDIIGDIRRAHC CCR5 (SEQ ID NO.14) SequenceIdentifiers (SEQ ID NO:1) CIRPNNNTRTSIRIGPGQAFYATGNIIGDIRQAYC (SEQ IDNO:2) CIRPNNNTRTSIRIGPRQAFYATGNIIGDIRQAYC (SEQ ID NO:3)CIRPNNNTRTSIRIGPGQAFYATGNIVGDIRQAYC (SEQ ID NO:4)RKSVHIGPGQAFYATGDIIGNIRKAHC (SEQ ID NO:5)CTRPNNNTRKSVHIGPGQAFYATGDIIGNIRKAHC (SEQ ID NO:6)CTRPNNNTRKSVHIGPGQAFYATGDIIGNIRQAHC (SEQ ID NO:7)CTRPNNNTRKSVHIGPGQAFYATGDIIGNMRKAHC (SEQ ID NO:8)CTRPINNRRKSIHMGPGQAFYGT.DDIIGDIRKARC (SEQ ID NO:9)CTRPSNNRRKSIHKGDQDKHSMEHDDVIGDIRKARC (SEQ ID NO:10)CTRPINNRRKSIHIGPGQAFYGT.DDIIGDIRQAHC (SEQ ID NO:11)CIRPNNNTRQSVHIGPGQALYTTEIIGDIRKAHC (SEQ ID NO:12)CTRPNNNTITSIRIGPGQAFYATGSIIGNIRQAHC (SEQ ID NO:13)CTRPNNNTITSIRIGPGQAFYATGSIIGNTRQAHC (SEQ ID NO:14)CTRPNDNIRKSVHIGPGQAFYATGDIIGDIRRAHC

Methods of env Gene Cloning

In cloning the env gene from patients by the use of long RT-PCR, twopotential problems may result: 1) recombination between molecules; and2) underestimates of sequence diversity. High fidelity cloning of thesamples above was achieved by routine performance of multiple RTreactions on limiting dilutions of RNA, followed by multiple PCR's oncDNAs obtained from each RT reaction. Performance of multiple PCR's oneach cDNA preparation increased the likelihood of amplifying a differentHIV-1 RNA species. These measures also decrease the chance ofrecombination. Accordingly, the following protocol was developed:

-   1. Peripheral blood was collected and separate into plasma and cell    components. Other fluids and tissues derived from an HIV-infected    individual can also be used, with minor modifications to the RNA    extraction protocol outlined below.-   2. HIV-1 RNA was quantitated in plasma by using NucliSens (Organon    Teknika Corp., Durham, N.C.), with a lower limit of quantitation set    at approximately 80 copies/ml.-   3. RNA extraction:    -   a) HIV-1 RNA was extracted from plasma using Qiagen's Viral RNA        Kit and following the manufacturer's standard protocol.    -   b) Samples were standardized by extracting a volume of plasma        equal to 10000 copies of HIV-1 RNA. For example, if the        patient's plasma viral load is 25000 copies/ml, 0.4 ml of plasma        in the extraction should be used.    -   c) Following extraction, the virus was resuspended in 100 ul of        Rnase-free water (to give a final concentration of <100 copies        of HIV-1 RNA per ul) and optionally treated with Rnase-free        Dnase to remove any contaminating DNA.-   4. RT-PCR using limiting dilution to ensure minority species    amplification:    -   a) Samples of serially diluted RNA template were generated in a        series of 1:5 dilutions using the following template        concentrations:        -   ˜100 copies/μl        -   ˜20 copies/μl        -   ˜4 copies/μl    -    This dilution series is sufficient to ensure minority species        amplification. Conditions are adaptable to achieve limiting        dilutions.    -   b) 1 ul aliquots of RNA template were distributed into the wells        of a PE2400 or PE9700 PCR tray-retainer and 8-24 tubes        containing of each RNA dilution were prepared. An example of the        template set-up for a PE2400 is shown in FIG. 3.    -   c) An RT reaction mix was prepared:

reagent per reaction Rnase-free H₂O 2 ul 10x PCRII buffer 2 ul 25 mMMgCl₂ 4 ul 10 mM dATP 2 ul 10 mM dCTP 2 ul 10 mM dGTP 2 ul 10 mM dTTP 2ul Rnase Inhibitor 1 ul 50 mM Random Hexamers 1 ul MMLV RT (50 U/ul) 1ul

-   -    All reagents are commercially available from Perkin Elmer. Each        well received a 19 ul aliquot. Samples were incubated for 60        minutes at 37° C., followed by heat inactivation for 5 minutes        at 95° C. Samples were stored at 4° C.    -   d) The Primary PCR reaction mix was prepared:

reagent per reaction sterile H₂O 67.5 ul 10x PCRII buffer 8 ul 25 mMMgCl₂ 2 ul primer HIVGao1F (20 uM) 1 ul primer HIVGao1R (20 uM) 1 ul Taqpolymerase (50 U/ul) 0.5 ul

-   -    Primer sequences for HIVGao1F and HIVGao1R were:

(SEQ ID NO:15) HIVGao1F: 5′-GGCTTAGGCATCTCCTATGGCAGGAAGAA-3′ (SEQ IDNO:16) HIVGao1R: 5′-GGCTTAGGCATCTCCTATGGCAGGAAGAA-3′

-   -    80 ul aliquots were transferred into each well containing the        RT mix. The cycle parameters were:

Cycle file Temp. Time 1 hold: 94° C. 5 minutes 5 cycles: 94° C. 1 minute50° C. 1 minute 72° C. 3.5 minute 30 cycles: 94° C. 1 minute 55° C. 1minute 72° C. 3.5 minute 1 hold: 72° C. 10 minutes 1 hold:  4° C. untilready for nested reaction

-   -   e) A nested PCR reaction mix was prepared:

reagent per reaction sterile H₂O 75.5 ul 10x PCRII buffer 10 ul 25 mMMgCl₂ 6 ul 10 mM dNTP blend 4 ul primer HIVGao2F (20 uM) 1 ul primerHIVGao2R (20 uM) 1 ul Taq polymerase (50 U/ul) 0.5 ul

-   -    Primer sequences for HIVGao2F and HIVGao2R are:

(SEQ ID NO:17) HIVGao2F: 5′-AGAAAGAGCAGAAGACAGTGGCAATGA-3′ (SEQ IDNO:18) HIVGao2R: 5′-AGCCCTTCCAGTCCCCCCTTTTCTTTTA-3′

-   -    Each well of a new PE2400 base received a 98 ul aliquot,        followed by 2 ul of each primary PCR reaction serving as a as        template for the nested PCR reaction. The same cycle parameters        as indicated for the primary PCR were applied.

-   5. Gel analysis of RT-PCR products:    -   a) 10 ul of each nested PCR product was run on a 1.5% agarose        gel.    -   b) Only the wells of the original RNA template dilution that        produced approximately 50% positive wells were cloned/sequenced        to ensure cloning/sequencing of an amplicion derived from a        single RNA template molecule. For example, if gel analysis        produced the following pattern based on the original RT layout,        only the 4 positive wells of the last row (the 1:25 or ˜4        copies/ul row) would be cloned/sequenced (FIG. 4.). All other        positives were discarded.    -   c) The chosen positives were either cloned or sent directly for        sequencing.

-   6. Cloning of RT-PCR products:    -   a) PCR reaction products were purified using Qiagen's Gel        Extraction Kit according to the manufacturer's standard        protocol.    -   b) Amplicons were cloned into Promega's pTarget Mammalian        Expression vector following a standard protocol, such as that        which is included with the pTarget Kit. Each selected positive        reaction was cloned once. In addition, only one clone from each        plate was picked/analyzed to ensure that the minority species        were fully represented    -   c) Plasmid DNA was prepared according to standard procedures for        ABI sequencing.

-   7. ABI sequencing of RT-PCR products or clones:    -   a) Standard automated sequencing on an ABI 370 series sequencing        machine was carried out. The following three primers were used        to ensure complete redundant sequencing of the V3 loop of the        envelope gene:

(SEQ ID NO:19) NL6942F: 5′-GCACAGTACAATGTACACATG-3′ (SEQ ID NO:20)NL7103F: 5′-ACAAGACCCAACAACAATACA-3′ (SEQ ID NO:21) NL7356R:5′-TGTATTGTTGTTGGGTCTTGT-3′

-   8. Sequence analysis:    -   a) The DNA sequence of the env V3 loop was determined.    -   b) Protein translation of the V3 loop was determined.    -   c) CCR5 or CXCR4 predictions were based on the scheme outlined        below:

-   -   d) The QXR value for the patient was calculated as:

QXR=(# of R5 clones)/(total # of clones)

Example 5 Qualitative HIV-1 Coreceptor Utilization Analysis using aHeteroduplex Tracking Assay (HTA) Specimen Accession and PlasmaPreparation

The purpose of this procedure is to describe the actions followed whenreceiving and preparing plasma specimens for HIV-1 coreceptorutilization analysis (QXR). Samples were removed from tubes in a steriledecontaminated hood. If lavender-top tubes of whole blood were sent, itwas centrifuged at room temperature for 10 minutes at 1,100×g (2300rpm). Tubes were removed from the centrifuge and checked for completeseparation. The plasma layer was transferred to freezer vials.

If, instead, frozen plasma had been shipped, it was either transferreddirectly to −80° C. freezer to be aliquoted and/or extracted at a latertime, or the plasma was thawed and transferred to appropriately labeledcroyogenic tubes in 200 μl aliquots, stored at −80° C. in RNA box, andentered into Sample Storage Log. If RNA was to be isolated on the sameday, 140 μl of plasma was transferred into a 1.5 ml screw-top conicalbase tube labeled with sample ID# and date. After the plasma had beenremoved, then the blood-draw tubes were discarded in appropriate wastecontainers for autoclaving.

Extraction of Viral RNA

The purpose of this procedure is to extract HIV-1 viral RNA from plasma.The extracted RNA is subsequently used for analysis of HIV-1 coreceptorutilization.

Plasma samples were thawed and equilibrated to room temperature. HIV-1RNA was extracted from plasma using Qiagen's Viral RNA Kit and followingthe manufacturer's standard protocol. All buffers including Lysis Buffer(AVL), Wash Buffer 1 (AW1), and Wash Buffer 2 (AW2) were preparedaccording to manufacturer's instructions. Any precipitate in buffers wasre-dissolved by heat incubation at 80° C. if necessary, but buffer wasallowed to re-equilibrate to room temperature before proceeding. Toavoid co-purification of cellular DNA, only cell-free body fluids shouldbe used for preparation of viral RNA. Samples that may contain cells(e.g., cerebrospinal fluid, urine, or swabs) should first be centrifugedfor 10 minutes at 2,000 rpm, and only the clarified supernatant used.

For patients samples with HIV-1 RNA loads<1.0×10⁵ copies/ml, the plasmavirions were pelleted by centrifuging the tubes for 90 minutes at10,000×g at 4° C. All tubes to be used were labeled with the correctidentifiers. 560 μl of Lysis Buffer (AVL) was pipetted into anappropriately labeled 1.5-ml screw-cap tube, then 140 μl plasma wasadded and mixed by pulse-vortexing for 15 seconds. Samples were lysedfor at least 10 minutes at room temperature (although samples may belysed for up to 24 hours at room temperature or 7 days at 4° C. withoutsignificant effect on the yield or quality of the purified RNA). The1.5-ml screw-cap tubes were briefly centrifuged (2-3 seconds at 8,000rpm) to remove drops from the inside of the lid. 560 μl of absoluteethanol was added and mixed by pulse-vortexing for 15 seconds. The1.5-ml screw-cap tubes were briefly centrifuged (2-3 seconds at 8,000rpm) to remove drops from the inside of the lid. 630 μl of the solutionwas carefully applied to an appropriately labeled QIAamp™ spin column.The sample or solution from the lysis tube was then carefully applied tothe column or tube by pipetting the sample into the tube without wettingthe rim or outside of the column. Tubes were centrifuged for 60 secondsat 6,000×g. The QIAamp™ spin columns were transferred into clean 2-mlcollection tubes. The supernatant-containing collection tubes werediscarded into a waste bucket. The remaining 630 μl of the solution wascarefully applied, without wetting the rim or outside of the column, toan appropriately labeled QIAamp™ spin column. The tubes were centrifugedfor 60 seconds at 6,000×g, and the QIAamp™ spin columns were transferredinto clean 2-ml collection tubes. The supernatant-containing collectiontubes were discarded into a waste bucket. The QIAamp™ spin columns wereopened carefully and 500 μl of Wash Buffer 1 (AW1) was added. Tubes werecentrifuged for 60 seconds at 6,000×g. The QIAamp™ spin columns weretransferred into clean 2-ml collection tubes. The supernatant-containingcollection tubes were discarded into a waste bucket. The QIAamp™ spincolumns were carefully opened and 500 μl of Wash Buffer 2 (AW2) wasadded. Tubes were centrifuged for 3 minutes at 10,000×g. The supernatantwas aspirated from the collection tubes using either a transfer pipettesor vacuum with trap. The pipette or tip was changed after eachaspiration. Tubes were centrifuged for 60 seconds at 6,000×g toeliminate any chance of wash buffer carryover. The QIAamp™ spin columnswere transferred into clean 1.5 ml microcentrifuge tubes. Thesupernatant-containing collection tubes were discarded into a wastebucket. 60 μl of Elution Buffer (AVE) was added to each column. Thepipette tip was changed for each tube. The columns were incubated for 60seconds at room temperature, followed by centrifugation for 60 secondsat 6,000×g. 10 μl of eluted ribonucleic acid was transferred into a new1.5-ml screw-cap tube for coreceptor utilization analysis. The remainingviral RNA (˜48-50 μl) was transferred into another 1.5-ml screw-cap tubefor long-term storage at ≦−70° C.

Reverse Transcription (RT) and Polymerase Chain Reaction (PCR)

The purpose of this procedure was to amplify a portion of the envelopegene of Human Immunodeficiency Virus type 1 (HIV-1), using viral RNAextracted from plasma as template. The resulting RT-PCR amplicon wassubsequently used for analysis of HIV-1 coreceptor utilization.

Two sets of PCR primers were used and are described in Table 3

TABLE 3 PCR primers First Set HTA6816F: of Primers 5′-CCT CAG CCA TTACAC AGG CCT GTC CAA AG-3′ (SEQ ID NO:35) HTA7359R: 5′-TTA CAG TAG AAAAAT TCC CCT C-3′ (SEQ ID NO:36) Second Set V3-7092F: of Primers 5′-GAATCT GTA GAA ATT AAT TGT ACA AGA C-3′ (SEQ ID NO:37) V3-7232R: 5′-TGC TCTACT AAT GTT ACA ATG TGC TTG TCT TAT-3′ (SEQ ID NO:38)

Reverse Transcriptase (RT) Master Mix Preparation:

GeneAmp RNA OCR core Kit reagents were thawed to room temperature,except for enzymes, which were removed from freezer only when needed.Reagents were mixed by vortexing and then microcentrifuged brieflybefore placing tubes in an ice bucket. A sterile 1.5 ml microcentrifugetube was placed in the ice bucket. Enough RT master mix was prepared toaccommodate the number of planned reactions plus one (to accommodatepipetting eror), based on the following amounts of reagents perreaction: 2 μl 10×RT-PCR Buffer II, 4 μl 25 mM MgCl, 2 μL 10 mM dCTP, 2μL 10 mM dGTP, 2 μL 10 mM dTTP, 2 μL 10 mM dATP, 1 μL 50 μM RandomHexamers, 1 μL Rnase Inhibitor (20 U/μl), 1 μL MuLV RT (50 U/μl). Mastermix and retainer assembly was transferred to a sterile laminar flowhood.

RNA Template Addition:

Patient RNA was thawed on ice followed by brief microcentrifugation toensure that all liquid is brought to the bottom. MicroAmp reaction tubeswere labeled and placed in retainer/tray assembly. RT master mix wasmixed by gently pipetting up and down a few times. 17 μl of master mixwas pipetted into each of the reaction tubes. 3 μl of viral RNAextracted from patient samples was added. One extraction positivecontrol (HIV-1 LAV) and one extraction negative control (Sera CarePlasma) were included with each RT-PCR run. Tubes were capped with capstrips and retainer/tray assembly was removed from the laminar flow hoodand transferred to thermocycler. The RT reaction mixtures were incubatedat 42° C. for 60 minutes followed by heat inactivation at 95° C. for 5minutes. The completed RT reaction can be stored at 4° C. (short-term)or −20° C. (long-term) until ready for cDNA amplification.

Primary PCR Master Mix Preparation:

GeneAmp RNA PCR Core Kit reagenets were thawed at room temperature,except for enzymes, which were removed from freezer only when needed.Reagents were mixed by vortexing and then briefly microcentrifuged andplaced in an ice bucket. A sterile 1.5 ml microcentrifuge tube wasplaced in the ice bucket. Enough cDNA amplification/primary PCR mastermix was prepared to accommodate the number of planned reactions plus one(to accommodate pipetting error), based on the following amounts ofreagents per reaction: 8 μL 10×PCR buffer II, 2 μL 25 mM MgCI₂, 1 μL ofeach primer (25 μM) (Table 3), 67.5 μL sterile water, and 0.5 μL Taqpolymerise (5U/μL). Primary PCR master mix and retainer assemblycontaining completed RT reactions were transferred to sterile laminarflow hood in template addition area.

cDNA Template Addition:

PCR master mix was mixed by gently gently pipetting up and down a fewtimes. 80 μL of master mix was overlayed into each of the RT reactiontubes, giving a total reaction volume of 100 μL. Tubes were capped withcap strips and retainer/tray assembly was removed from laminar flow hoodand transferred to a thermocycler, which was programmed for cDNAamplification as follows: PCR mixtures were pre-incubated at 94° C. for5 minutes, followed by 35 cycles of three-step incubations at 94° C. for15 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute, followed bya 5 minute incubation at 72° C. The completed primary PCR reaction wasstored at 4° C. (short-term) or −20° C. (long-term) until ready fornested amplification.

Secondary/Nested PCR Master Mix Preparation:

GeneAmp RNA PCR Core Kit reagents were thawed at room temperature,except for enzymes, which were removed from freezer only when needed.Reagents were vortexed to mix and then briefly microcentrifuged andplaced in an ice bucket. A sterile 1.5 ml microcentrifuge tube wasplaced in the ice bucket. Enough cDNA amplification/primary PCR mastermix was prepared to accommodate the number of planned reactions plus one(to accommodate pipetting error), based on the following amounts ofreagents per reaction: 10 μL 10×PCR Buffer II, 6 μL 25 mM MgCl₂, 4 μL 10mM dNTP blend, 1 μL of each secondary primer (25 μM)(Table 3), 75.5 μLsterile water, 0.5 μL Taq polymerase (5U/μL). Primary PCR master mix andretainer assembly containing completed RT reactions were transferred tosterile laminar flow hood in the template addition area.

Secondary/Nested PCR Template Addition:

MicroAmp reaction tubes were labeled and placed in retainer/trayassembly. Secondary/nested PCR master mix was mixed by gently pipettingup and down a few times. 98 μL of master mix was added into each of thereaction tubes. 2 μL of the primary PCR reaction was added tocorresponding secondary PCR reaction tube for a total volume of 100 μL.Tubes were capped with cap strips and the retainer/tray assembly wasremoved from the laminar flow hood and transferred to a thermocyclerwhich was programmed for cDNA amplification as follows: re-incubated at94° C. for 5 minutes, followed by 35 cycles of three-step incubations at94° C. for 15 seconds, 55° C. for 30 seconds, 72° C. for 1 minute,followed by a 5 minute incubation at 72° C. The completed primary PCRreaction was stored at 4° C. (short-term) or −20° C. (long-term) untilready for nested amplification.

Sample Preparation for Agarose Gel Analysis:

6× gel-loading buffer was prepared as follows: 0.25% bromophenol blue,0.25% xylene cyanol, 30% glycerol, and water up to desired final volume.A stock solution can be prepared and stored at room temperature. 20 μLof 6× gel-loading buffer was added to each secondary/nested PCR reactiontube. Samples were mixed by pipetting up and down.

Agarose Gel Preparation:

5×-TBE buffer was diluted to 0.5× with distilled water. Ethidium bromidewas added to a final concentration of 0.5 μg/ml. A 4% (w/v) GTG NuSieveagarose solution was prepared by adding 6 g agarose to 150 ml0.5×TBE/EtBr in a 250 ml glass Erlenmeyer flask. The agarose/TBEsolution was gently mixed for 10 minutes at room temperature (to allowthe agarose to hydrate), followed by heating in the microwave at 40%power for 10 minutes, mixing occasionally, until all agarose iscompletely dissolved. The dissolved agarose solution was gently cooledunder cold running water and then poured into a previously set-up geltray (with appropriate size gel comb), while making sure to minimizebubbles. The agarose was allowed to completely solidify forapproximately 30-60 minutes at room temperature.

Agarose Gel Electrophoresis:

Once the agarose solidified, the comb was gently removed and the gelapparatus was prepared to receive running buffer. 0.5×TBE buffer,containing 0.5 μg/ml ethidium bromide, was slowly poured into theelectrophoresis rig until the gel was completely submerged. 10 μl of the100-bp DNA ladder was loaded into the first well of the agarose gel.Each secondary/nested PCR sample was loaded into subsequent wells of theagarose gel. The lid was placed on the gel apparatus and the voltage wasturned on at 100-200V constant current until the bromophenol blue (lowerdye front) reached the end of the gel. Care was taken not to run the gelto long so that the samples were not lost. The gel was visualized on theanalytical setting of the UV transilluminator and photographed forrecord-keeping purposes. The desired PCR amplicon was approximately140-bp in size.

DNA Extraction:

The gel was visualized using the preparative setting on the UVtransilluminator. Each sample band was cut out of the gel with a cleanrazor blade or scalpel and place in a pre-weighed 1.5 ml microcentrifugetube. The band was cut as close to its edges as possible, in preparationfor the QIAquick separation kit which allows for a maximum of 400 μg ofagarose. Blades were changed between bands to avoid samplecross-contamination. Amplified DNA was extracted from each agarose sliceusing Qiagen's QIAquick separation protocol (e.g. Qiagen's QIAquick GelExtraction Kit Protocol (03/2001 Handbook)).

Purified DNA was analyzed spectrophotometrically and was adjusted to˜250 ng/μL. Approximately 90 μL DNA was used for the subsequentcoreceptor analysis procedures. The purified DNA was transferred tosterile 1.5 ml screw-cap tubes and was either stored at 4° C.(short-term) or −20° C. (long-term) until ready for HTA analysis or TOPOTA cloning.

Polymerase Chain Reaction (RT-PCR) Amplication of Cloned HIV-1 Sequencesto Generate Fluorescently-Labeled Probes for Qualitative andQuantitative Coreceptor Utilization Analysis

The purpose of this procedure was to amplify a portion of the envelopegene of Human Immunodeficiency Virus type 1 (HIV-1), using clonedplasmid DNA. Fluorescent-labeled PCR primers were used to generatefluorescein-conjugated DNA probes. The resulting probes weresubsequently used for qualitative and quantitative analysis of HIV-1coreceptor utilization. Two sets of fluorescently-labeled primers wereused to generate fluorescein-conjugated DNA probes, with the forwardprimer of each pair covalently linked at the 5′ end to fluorescein. Forprimers see Table 4.

TABLE 4 Primers to make probes 5′F*V3-7092F: 5′-/56-FAM/ GAA TCT GTA GAAATT AAT TGT ACA AGA C-3′ V3-7232R: 5′-TGC TCT ACT AAT GTT ACA ATG TGCTTG TCT TAT-3′ 5′F*V3HTA-EcoRI-F: 5′-/56-FAM/ AAT TCG CCC TTG AAT CTGTAG AAA TTA AT-3′ V3HTA-EcoRI-R: 5′-AAT TCG CCC TTT TIT GCT CTA CTAATG-3′

PCR Master Mix Preparation to Generate Probe for Qualitative HTA:

GeneAmp RNA PCR Core Kit reagents were thawed at room temperature,except for enzymes, which were removed from freezer only when needed.Reagents were vortexed to mix and then briefly microcentrifuged andplaced in an ice bucket. A sterile 1.5 ml microcentrifuge tube wasplaced in the ice bucket. Enough cDNA amplification/primary PCR mastermix was prepared to accommodate the number of planned reactions plus one(to accommodate pipeting error), based on the amounts of reagents perreaction as follows: 10 μL 10×PCR buffer II, 6 μL 25 mM MgCl₂, 4 μL 10mM dNTP blend, 1 μL of each primer to make probe (at 25 μM)(Table 4),76.5 μL sterile water, 0.5 μL Taq polymerase (5U/μL). At least fourreactions were planned (one for each probe). A negative controlcontaining sterile water instead of plasmid DNA was also prepared. This“qualitative” PCR master mix and retainer tray assembly were transferredto a sterile laminar flow hood in the template addition area.

PCR Master Mix Preparation to Generate Probe for Quantitative HTA:

GeneAmp RNA PCR Core Kit reagents were thawed at room temperature,except for enzymes, which were removed from freezer only when needed.Reagents were vortexed to mix and then briefly microcentrifuged andplaced in an ice bucket. A sterile 1.5 ml microcentrifuge tube wasplaced in the ice bucket. Enough cDNA amplification/primary PCR mastermix was prepared to accommodate the number of planned reactions plus one(to accommodate pipeting error), based on the amounts of reagents perreaction as follows: 10 μL 10×PCR buffer II, 6 μL 25 mM MgCl₂, 4 μL 10mM dNTP blend, 1 μL of each primer to make probe (at 25 μM)(Table 4),76.5 μL sterile water, 0.5 μL Taq polymerase (5U/μL). At least fourreactions were planned (one for each probe). A negative controlcontaining sterile water instead of plasmid DNA was also prepared. This“quantitative” PCR master mix and retainer tray assembly weretransferred to a sterile laminar flow hood in the template additionarea.

PCR Template Addition:

MicroAmp reaction tubes were labeled and placed in retainer/trayassembly. Each of the “qualitative” and “quantitative” PCR master mixeswere mixed by gently pipetting up and down a few times. 99 μL of eachmaster mix were added into reaction tubes. 1 μL of each plasmid DNAtemplate (SF₁₆₂, JR-CSF, Sw54, and Sw87; derived from primary HIV-1strains of the same name) was added to corresponding PCR reaction tubefor a total volume of 100 μL. Tubes were capped with cap strips andretainer/tray assembly was removed from laminar flow hood andtransferred to thermocycler, which was programmed for cDNA amplificationas follows: pre-incubation at 94° C. for 5 minutes, followed by 35cycles of three-step incubations at 94° C. for 15 seconds, 55° C. for 30seconds, 72° C. for 1 minute, followed by a 5 minute incubation at 72°C. The completed PCR reaction can be stored at 4° C. (short-term) or−20° C. (long-term) until ready for cDNA amplification. PCR productswere analyzed and gel-purified on a 4% agarose gel as described above.

Qualitative HIV-1 Coreceptor Utilization Analysis using a HeteroduplexTracking Assay (HTA)

This assay uses a heteroduplex tracking (HTA) technique to analyze aportion of the Human Immunodeficiency Virus type 1 (HIV-1) envelope geneencompassing the key determinates of coreceptor utilization. Sequencedifference between CCR5- and CXCR4-using variants result in distinctheteroduplex electrophoretic mobilities that allow the overall numberand relative proportion of distinct variants to be estimated, even insamples consisting of heterogeneous CCR5 and CXCR4 pools. Plasmaspecimens showing heteroduplex patterns indicative of CXCR4 strains arethen subjected to further analysis to quantitate the portion of CCR5 andCXCR4 viruses in the patient quasispecies. Interpretation of the gels isbased on the banding pattern seen in each gel lane. The absence ofclearly distinct X4-heteroduplex bands is indicative of a predominanceof CCR5-utilizing strains of HIV-1. A schematic representation ofqualitative HTA analysis of four different of targets: probe only, aCCR5 virus V3 region of the envelope gene a CXCR4 virus V3 region of theenvelope gene, and a heterogeneous mix of CCR5 and CXCR4 virus V3regions, is shown in FIG. 6.

Preparation of Non-Denaturing Polyacrylamide Gels:

12% acrylamide solution was prepared to accommodate the number ofplanned gels, based on the following amounts of reagents per 75 mL gel:22.5 mL 40% (29:1) acrylamide/bis-acrylamide stock solution, 36.9 mLdeionized water, 15 mL 5× Tris-Borate-EDTA (TBE) stock buffer, 52.5 μLTEMED, 525 μL 10% AMPS, freshly prepared in deionized water. Thereservoirs of the electrophoresis tank were filled with 1×TBE (made with1 part 5×TBE and 4 parts deionized water).

Heteroduplex Formation:

If necessary, prepared probe and target DNA were thawed at roomtemperature. Probe and target DNA were vortexed to mix and thenmicrocentrifuged briefly and placed in an ice bucket. Between two andfour sterile 1.5 ml microcentrifuge tubes were placed in the ice bucket(one tube per probe). Enough HTA annealing mix was prepared toaccommodate the number of planned reactions plus one (to accommodatepipeting error), based on the following amounts of reagents perreaction: 3 μL 10×HTA annealing buffer, 5 μL FITC-labeled probe, 2 μLsterile water. MicroAmp reaction tubes were labeled and placed inretainer/tray assembly. HTA annealing mix was mixed by gently pipetingup and down a few times. 10 μL of master mix were aliquotted into eachof the reaction tubes. 20 μL of viral RNA extracted from patient sampleswas then added. There were two to four reactions for each patientsample—one for each probe used. One positive control (the purified HIV-1LAV extraction control) and one negative control (water only) wereincluded in each run. These controls were also used to determine theamount of homoduplex and heteroduplex DNA present in each experiment.Tubes were capped with cap strips and the retainer/tray assembly wasremoved from the laminar flow hood and transferred to a thermocycler.The HTA annealing reaction was run for 2 minutes at 94° C., followed byquenching to 4° C. (short-term). The resulting reactions were placed onice and immediately loaded on a 12% non-denaturing polyacrylamide gel.

Polyacrylamide Gel Electrophoresis:

6× gel-loading buffer was prepared by combining: 0.25% bromophenol blue,0.25% xylene cyanol, 30% glycerol, water up to desired final volume. Astock solution may be prepared and stored at room temperature. 6 μL of6× gel-loading buffer was added to each HTA annealing reaction tube andwas mixed by pipeting up and down. Using a sequencing gel loading tip,the entire HTA annealing reaction was gently loaded into thepolyacrylamide gel wells. The electrodes were connected to the powersupply and the gel was run at a constant voltage until the last of theupper xylene cyanol dye front runs off the bottom of the gel(approximately 6 hours at 250V or overnight at 90V), or until thepolyacrylamide gel marker dyes had migrated the desired distance.

Scanning and Gel Analysis:

The FluorImager 595 controls were adjusted to the following settings: 1)single label dye; 2) 488 nm excitation; 3) no emission filter; 4) nocalibration; 5) 1000V PMT; 6) high sensitivity; 7) 200 μm pixels; and 8)16-bit resolution. ImageQuaNT™ software package was used to display thegel image, once the scan was complete.

Interpretation of the gels is based on the banding pattern seen in eachgel lane. The absence of clearly distinct heteroduplex bands isindicative of a predominance of CCR5-utilizing strains of HIV-1. Patientsamples that contain only CCR5 viruses were assigned a QXR value of 1.0[where QXR=(number of CCR5 clones)/(total number of clones analyzed)].Specimens with detectible CXCR4 virus, on the other hand, were subjectedto further quantitative analysis to quantitate the portion of CCR5 andCXCR4 viruses in the patient quasispecies using the procedures outlinedbelow.

Example 6 Quantitative Coreceptor Utilization Analysis using aHeteroduplex Tracking Assay (HTA) Cloning of HIV-1 Envelope Sequencesfor Quantitative Coreceptor Utilization Analysis:

The purpose of this procedure was to clone HIV-1 envelope sequences forquantitative coreceptor utilization anaylsis. HIV-1 RNA isolated andamplified from patient plasma was cloned into a plasmid vector(pCR®2.1-TOPO®, Invitrogen), used to transform chemically competentEscherichia coli, and plated onto selective bacterial media.

The following reagents per reaction were gently mixed and incubated for5 minutes at room temperature and then placed on ice: 4 μL extracted DNAamplicon/sterile water, 1 μL salt solution, and 1 μL TOPO™ vector.Enough OneShot® E. coli cells were thawed on ice to accommodate thenumber of planned cloning reactions. 2 μL of the TOPO® cloning reactionwas added to a vial of OneShot® E. coli and mixed gently using a pipettetip. E. coli was incubated on ice for 30 minutes and heat-shocked for 30seconds at 42° C. 250 μL of room temperature SOC medium was added to thecells. Tubes were capped and incubated at 37° C. with gentle shaking for1 hour. The entire transformation mixture was spread onLB/ampicillin/X-gal plates. The number of blue and white colonies oneach plate were counted and recorded.

Isolation, Preparation, and Screening of Plasmid DNA Encoding ClonedHIV-1 Envelope Sequences for Quantitative Coreceptor UtilizationAnalysis

The purpose of this procedure was to prepare high quality plasmid DNAencoding cloned HIV-1 envelope sequences for quantitative coreceptorutilization analysis. HIV-1 RNA isolated and amplified from patientplasma was cloned into a plasmid vector, grown overnight in 1-3 mL ofEscherichia coli bacterial culture, and purified using a commerciallyavailable plasmid miniprep kit (Perfectprep®, Eppendorf, Westbury,N.Y.). Analysis for the viral specific sequences was carried out bydigestion of the recombinant plasmid with restriction enzyme EcoRI (20U/μL).

Screening of Plasmids by Restriction Enzyme Digestion:

A sterile 1.5 ml microcentrifuge tube was also placed in the ice bucket.Digests were performed in duplicate (one digest for agarose gel analysisand one digest for quantitative HTA analysis). Tubes were capped withstrips and transferred to the thermocycler, which was programmed forEcoRI digestions as follows: 37° C. for 37 minutes followed by 95° C.for 1 minute. The completed restriction enzyme digests can be stored at4° C. (short-term) or −20° C. (long-term) until ready for gel and HTAanalysis.

10 μl of the 100-bp DNA ladder was loaded into the first well of theagarose gel. Each secondary/nested PCR sample was loaded into subsequentwells of the agarose gel. The desired band was approximately 160 bp insize. An additional band, representing linearized TOPO TA vector wasalso seen. The coreceptor utilization profile of positive transformantswas then analyzed by HTA.

Quantitative Analysis Using an HTA of HIV-1 Coreceptor Utilization

This assay uses the heteroduplex tracking (HTA) technique described inExample 1 to analyze a portion of the Human Immunodeficiency Virus type1 (HIV-1) envelope gene encompassing the key determinates of coreceptorutilization. Individual clones from patient plasma specimens whichshowed heteroduplex patterns indicative of CXCR4 strains were subjectedto analysis to accurately quantitate the portion of CCR5 and CXCR4viruses in the patient quasispecies. DNA heteroduplex tracking analysiswas performed with the coreceptor utilization profile of a minimum oftwenty positive transformants from each patient sample determined byusing two probes to screen each clone. Probes were prepared from onelaboratory CCR5 isolate (SF162 or JR-CSF) and one primary CCR5 isolate(Sw54 or Sw87). The QXR value for each patient specimen was thencalculated based on the number of CCR5-specific clones obtained fromeach sample as follows: QXR=(number of CCR5 clones)/(total number ofclones analyzed). FIG. 6 is a schematic representation of HTA analysisof four different targets: probe only, a CCR5 virus V3 region, a CXCR4virus V3 region, and mixed quasispecies containing both CCR5 and CXCR4virus V3 regions.

Example 7 Validation Experiments PCR Primer Design

A common problem is low or no target DNA yield following PCR, reflectingeither PCR efficiency or sample preparation problems. This problem wasalleviated in part by use of a commercially available RNA extraction kit(Qiagen Viral RNA Kit), and in part by use of a small amount of pooledHIV-1 LAV, which is always simultaneously extracted as a positive RNAcontrol. This practice is part of the standard operating procedure.

Primers used herein were designed to match the lade B consensus sequenceas posted on the Los Alamos National Laboratories HIV database. Usingthis primer set, inventors currently have a success rate of 98.4% inamplifying envelope sequences from patient samples with a viral load ofat least 1000 copies per milliliter of plasma.

Variant Sampling

Correct sampling is a recurring and frequently overlooked potentialproblem in subcloning and sequencing analyses of complex populations.Previously, inventors circumvented this problem by sequencing subclonesderived from multiple independent PCR's or sequencing the dilution endpoint directly. For genetic differences in quasispecies detected aschanges in HTA patterns to be significant, the populations beingcompared must be appropriately sampled. Any claims of quasispecieschanges using HTA or other methods of direct population analyses must besubstantiated through reproducibility of the results using the productof duplicate, independent amplifications to document proper sampling. Toensure proper variant sampling using the technique, inventors havecompared the HTA results from independent duplicate PCR's. Inventors rana series of HTA's using different amounts of input template and multipleparallel amplifications to prove that inventors can consistently amplifyall of the majority and minority variants in a patient sample. Threelevels of sequence difference between target DNA mixtures were selectedto span the diversity found in the HIV-1 envelope gene. Duplicate10-fold serial dilutions of viral RNA were then amplified by PCR andanalyzed by HTA using our various probes. Inventors saw identical HTApatterns in each independent PCR, indicating reproducible and thereforecorrect sampling of the target populations. These studies also wererepeated using various biological and molecular clones derived fromprimary isolates from patients previously examined in our treatmentstudy (1). Finally, inventors repeated these sampling studies using RNAfrom primary patient isolates of known and unknown coreceptor usage. Ineach case, analysis of duplicate, independent PCR's demonstrated thatthe primers and the optimized PCR reaction conditions reproduciblyamplify a mixture of HIV-1 variants that adequately reflects thepopulation in the original sample.

Limits of Detection

The main advantage of HTA is its ability to simultaneously analyzemultiple genetic variants coamplified by PCR. Using optimized reactionconditions, HTA's can be used to detect variants that represent lessthan 1% of the total quasispecies population (5). The ability of thecoreceptor-specific HTA to detect rare variants has been examined byreconstituting mixtures of virus using laboratory isolates with knowncoreceptor usage. The sensitivity of the HTA method to detect R5 and X4isolates was independently ascertained by using reconstituted sampleswith QXR values at or near 0 and 1, respectively. These experiments havedemonstrated that inventors can routinely and reproducibly detect CCR5and CXCR4 variants that represent as little as 0.2% of the total viralpopulation.

Assay Validation Using Patient Isolates

Over the last five years inventors have isolated a large number ofbiologic and molecular clones of HIV-1, allowing us to compare genotypicpredictions of coreceptor usage (either by performing V3 loop sequencingor by using our HTA method) with phenotypically-determined coreceptorpreference.

Previously, inventors used a rapid RT-PCR based genotypic method tomeasure QXR, the proportion of HIV-1 utilizing CCR5 or CXCR4 as acoreceptor. This method relied on sequence analysis of the V3 region ofthe HIV-1 envelope gene to determine QXR. A total of 424phenotypically-characterized biological and molecular clones of HIV-1were analyzed, yielding the following data:

Phenotypic Result CCR5-using CXCR4-using Genotypic Prediction CCR5-using225 8 by V3 Sequencing CXCR4-using 12 179For detection of CCR5 strains of HIV-1, this sequencing-based approachthus achieves 94.9% sensitivity and 95.7% specificity.

A subset of clones from this sample set has also been examined using thenewer HTA approach. A total of 392 clones have been analyzed:

Phenotypic Result CCR5-usin CXCR4-using Genotypic Prediction CCR5-using232 0 by HTA CXCR4-using 3 157For detection of CCR5 strains of HIV-1, the HTA method achieves ˜100%sensitivity and specificity. Conversely, for detection of CXCR4 strainsof HIV-1, this method attains ˜100% sensitivity and 98.7% specificity.The predictive values for detecting CCR5 and CXCR4 strains are 100% and98.1%, respectively.

Example 8 HIV-1 Coreceptor Usage and CXCR4-Specific Viral Load PredictClinical Disease Progression

The purpose of this example is to show the relationship of HIV-1coreceptor usage to clinical endpoints, and in particular theidentification of patients at high risk for AIDS or death before orduring cART.

Pioneering cohort studies of viral phenotype were performed before cARTwas introduced, and measured a phenotypic characteristic of X4 viruses,induction of syncytia in tissue culture (Koot et al. (1993)). Althoughthe presence of syncytia-inducing virus was a strong predictor of HIV-1disease progression, cell culture-based syncytia assays were impracticalfor clinical use and remained as research tools.

cART has become so effective that relatively few treated patientsexperience disease progression, and clinical trials rely primarily onsurrogate markers for assessment (Ledergerber et al. (1999); and Mocroftet al. (2003)). Previous studies revealed, however, that patientsdisplayed clinical benefits from therapy beyond those mediated throughchanges in CD4 count and HIV-1 load (Mocroft et al. (2003); Ledergerberet al. (2004); and Miller et al. (2004)). Inventors therefore focused onthe relationship of HIV-1 coreceptor usage to clinical endpoints, askingwhether quantification of coreceptor usage identified patients at highrisk for AIDS or death during cART. To quantify HIV-1 coreceptor usageand determine X4-specific HIV-1 load, inventors developed a sensitive,nucleic acid-based assay to determine the proportion of virus in apatient's plasma that uses each coreceptor.

By examining patients in the Swiss HIV Cohort Study (SHCS) (Ledergerberet al. (1999); and Ledergerber et al. (1994) Soz Praventivmed39:387-94), inventors assessed the predictive value of HIV-1 coreceptorusage before the initiation of therapy and, in those with persistentviraemia during cART, after 6 months of treatment.

The SHCS is a prospective, clinic-based, observational study ofHIV-1-infected adults initiated in 1988, with documentation of follow-upvisits every six months (Ledergerber et al. (1994)). A subset ofpatients were selected from 2674 who initiated cART between 1995 and1998 and who were described in our previous report on clinicalprogression and persistent viremia (Ledergerber et al. (1999)). Thestudy was approved by Institutional Review Boards at each site and eachpatient signed informed consent.

Selection of Study Subjects and Samples

First, inventors identified the 170 patients who subsequently progressedto a new clinical AIDS-defining event or death while receiving cART. Toqualify for the present study, patients needed sufficient plasmaavailable from the SHCS visit preceding the initiation of cART, calledbaseline, and an HIV-1 load≧1000 copies/mL at that visit. The medianinterval between the initiation of cART and the baseline visit was 18days [Interquartile range (IQR) of −64-0 days]. Follow-up samples wereobtained after ˜6 months of cART, with a median interval between thepre- and post-cART samples of 184 days (IQR of 135-212 days). Because anHIV-1 load≧500 copies/mL was required of post-cART specimens, follow-upsamples were analysed only in patients who did not achieve completevirologic suppression. Selection of all specimens allowed for at leastone additional contemporaneous aliquot remaining in stock for futureprojects. Inventors retrieved 115 baseline specimens from progressors,and 19 of these from one site could not be analysed owing to a problemin shipping and handling. Inventors therefore quantified coreceptorusage in 96 baseline samples. Paired follow-up specimens were availablefrom 39 patients, with coreceptor results obtained from all 39.

As a second step, inventors identified pre- and post-cART aliquots from91 patients who did not progress within the period of the original study(up to Dec. 31, 1998) and who were matched to progressors according tothe clinic site and year cART was initiated. With the requirement forone aliquot remaining in stock, 4 specimens lost to handling, and ourinability to amplify from 7, inventors quantified coreceptor usage in 84baseline and 31 follow-up samples from non-progressors. In total,inventors analysed 180 baseline and 70 follow-up samples.

Markers of Disease Progression

CD4 lymphocyte counts were measured by using flow cytometry and HIV-1RNA levels, by using the Cobas Amplicor test, with a level of detectionof 500 copies/mL (Roche Diagnostics, Rotkreuz, Switzerland) (Ledergerberet al. (1999)).

Quantification of HIV-1 Coreceptor Usage

Inventors quantified the proportion of HIV-1 variants using R5 or X4 ineach plasma sample by employing a non-radioactive, DNA heteroduplextracking assay (HTA) developed based upon previous methods (Delwart etal. (1997) Methods 12:348-54); and Nelson et al. (1997) J. Virol.71:8750-8). Because X4 variants ordinarily coexist in a viral swarmalong with R5, (Berger et al. (1998); Shankarappa et al. (1999);Scarlatti et al. (1997); Koot et al. (1993); and Connor et al. (1997)),it was necessary to quantify the proportion of viruses in plasma usingeach coreceptor. This proportion was expressed as a variable called theQuantity of X4 and R5 (QXR), which represents the fraction of virus in aspecimen using the R5 coreceptor. If QXR=1, almost all of the viruses ina population use R5; if QXR=O, almost all use X4. If a mixture of R5 andX4 viruses are present, QXR<1 (Philpott et al. (2001)).

Because the key determinants of viral coreceptor usage are encoded bythe third variable domain (V3) of the envelope gene, (Ho et al. (2005)J. Virol. 79:12, 296-303; and Pastore et al. (2006) J. Virol. 80:750-8),inventors developed a nucleic acid-based assay focusing on this regionof the HIV-1 genome. Viral RNA was extracted from patient samples byusing a QIAamp viral RNA extraction kit (Qiagen, Valencia, Calif.), withsamples from different patients processed separately to minimisepossible cross-contamination or mislabeling. Reverse transcription andPCR amplification (RT-PCR) of a 143 by fragment spanning the V3 regionof the env gene was performed as described under conditions designed tooptimise efficiency and variant sampling (Philpott et al. (2001); andFang et al. (2003) AIDS 18:153-159).

DNA heteroduplex formation was carried out by annealingfluorescein-labeled probes derived from four CCR5-using HIV-1 strainswith a 10-fold excess of unlabeled target DNA. Sequence differencesbetween envelope variants resulted in distinct heteroduplexelectrophoretic mobilities, allowing rapid estimation of the overallnumber and relative proportion of R5 and X4 variants.

Validation Experiments

To evaluate whether the observed heteroduplex banding patternsaccurately predicted coreceptor usage, inventors used the HTA tocharacterize ˜400 biologic and molecular HIV-1 clones of knowncoreceptor specificity. The predictive value of the HTA method fordetecting R5 and X4 strains was 100% and 98.7%, respectively. Thesensitivity of the HTA method also allows rare variants to be detectedand quantified; HIV-1 subpopulations that represent as little as 1% ofthe total quasispecies be can readily identified (Delwart et al. (1997).Those samples harbouring X4 strains (QXR<1) were subjected to moredetailed analysis, during which V3 loops were cloned and individuallyanalysed by using HTA.

After determining the coreceptor usage of each clone, inventors thencalculated QXR for each plasma specimen by applying a mathematical modelderived previously (Philpott et al. (2001). The X4-specific HIV-1 loadwas calculated by multiplying the total viral load by the proportion ofthe viral population using

X4: X4-specific viral load=(total HIV-1 load)(1−QXR)

Analyses of these and other plasma samples demonstrated that inventorswere capable of determining HIV-1 coreceptor usage in 97% of sampleswith HIV-1 RNA loads≧1000 copies/mL and 85% of those with viralloads<1000 copies/mL.

Statistical Analysis

Virologic responses were measured in terms of the percentage of patientswith HIV-1 RNA<500 copies/mL six months after initiating cART. Forimmunologic responses, inventors determined the change in CD4 countsbetween values obtained at baseline and those obtained at the visitclosest to six months. QXR, the proportion of plasma HIV-1 using CCR5,was stratified into two categories:

(1) QXR=1 if all virus identified uses CCR5, and

(2) QXR<1 if X4 virus is detected

The association between virologic responses and baseline QXR wasassessed by comparing the percentages of patients with undetectableHIV-1 RNA load across the different strata by using Fisher's exact test.Immunologic responses across two strata were compared by Wilcoxonrank-sum tests.

Kaplan-Meier curves and Cox proportional hazard regression models wereapplied to quantify the association of baseline or follow-up QXR (equal1 vs. less than 1) with subsequent clinical progression, defined as anew clinical AIDS-defining event or death.

In addition to the two QXR strata, inventors included an additionalmodel analysing the relationship of X4 viral load to HIV-1 diseaseprogression by stratifying X4-specific viral load into three strata:

(1) patients without detectable X4-specific viral load (i.e., QXR=1)

(2) patients with detectable X4 viraemia below the median value ofX4-specific viral loads, and

(3) patients with detectable X4 viraemia above the median value ofX4-specific viral loads.

To compare the predictive capacity with the established progressionmarkers CD4 and HIV-1 RNA load inventors included concurrent log₂transformed CD4 values and log₁₀ transformed HIV-1 loads in theunivariable and multivariable Cox models. Inventors applied inverseprobability weights to adjust for sampling bias.

Inventors used STATA (Version 9.1, StataCorp, College Station, Tex.) foranalyses.

QXR can Predict the Response to cART

To examine whether QXR can predict the response to cART, inventorsstudied a subset of SHCS patients who initiated treatment in 1995-1998.Inventors compared 96 patients who progressed to a clinicalAIDS-defining event or death with 84 contemporaneous non-progressors.Baseline demographic characteristics showed that progressing andnon-progressing patients were comparable in age, sex, and risk for HIV-1acquisition (P>0.1) (Table 5).

TABLE 5 Characteristics of 180 patients at initiation of cART(baseline). Value* Progressors Non-progressors Characteristic n = 96 n =84 Total Median (IQR^(†)) age, years 36 (32-43) 35 (30-41) 36 (31-42)Sex Male 65 (68%) 56 (67%) 121 (67%) Female 31 (32%) 28 (33%) 59 (33%)Risk factor for HIV-1 acquisition Injection drug use 38 (40%) 27 (32%)65 (36%) Male homosexual contact 27 (28%) 32 (38%) 59 (33%) Heterosexualcontact 27 (28%) 25 (30%) 52 (29%) Other or unknown 4 (4%) 0 (0%) 4 (2%)Clinical stage CDC stage A 20 (21%) 30 (36%) 50 (28%) CDC stage B 36(37%) 20 (24%) 56 (31%) CDC stage C 40 (42%) 34 (40%) 74 (41%) Median(IQR) CD4*cell count per μL 50 (18-137) 119 (57-291) 90 (29-192) Median(IQR) viral load, log₁₀ copies/mL 5.3 (4.6-5.6) 4.5 (4.0-5.2) 4.9(4.2-5.4) Treatment naïve when starting cART 37 (39%) 38 (45%) 75 (42%)QXR Equals I 52 (54%) 67 (80%) 119 (66%) Less than 1 44 (46%) 17 (20%)61 (34%) Mean (IQR) QXR 0.85 (0.8-1.0) 0.92 (1.0-1.0) 0.88 (0.9-1.0)X4-specific viral load, log₁₀ copies/mL^(±) 0 (QXR = 1) 52 (54%) 67(80%) 119 (66%) 2.2-4.3 17 (18%) 13 (15%) 30 (17%) >4.3 27 (28%) 4 (5%)31 (17%) Mean (IQR) X4 viral load, logo copies/mL 3.5 (2.6-4.4) 2.8(2.6-2.6) 3.2 (2.6-3.7) *Number of patients unless otherwise stated.^(†)IQR: lnterquartile range. ^(±)Stratification according to median of61 values with non-zero values of X4-specific viral load.As expected, however, the progressors exhibited evidence of moreadvanced HIV-1 infection (Table 5). Not only did they display lower CD4counts and higher HIV-1 loads than did non-progressors, but they alsowere more likely to harbour X4-specific HIV-1 variants (P<0.0001 for allthree comparisons). HIV-1 coreceptor usage is expressed here as a QXRvalue, with QXR<1 signifying a mixture of R5 and X4 variants and QXR=1signifying all R5 strains. A significantly larger proportion ofprogressors exhibited QXR<1 than did non-progressors, and the meanX4-specific HIV-1 load was therefore higher in progressors as well(P<0.0001).

Patients whose samples were analyzed in this study were comparable tothe entire SHCS population with respect to gender, age, and mode ofHIV-1 acquisition (all P>0.1). Among SHCS non-progressors, however,individuals whose samples were analysed for QXR exhibited more advancedimmunosuppression than patients whose samples were not analysed; 40% vs.24% had CDC stage C disease, with a median baseline CD4 cell count of119 vs. 207 cells/μL (both P<0.01). Among progressors, 42% of patientswith QXR results had reached CDC stage C and the median baseline CD4count was 50 cells/μL.

Inventors do not have an obvious explanation for this imbalance, butbecause it diminishes the difference between baseline predictorsobserved in progressors and non-progressors, it will result in anunderestimation of the true effect of QXR.

Association of QXR with Immunologic and Virologic Responses

Inventors first determined whether QXR values before and duringtreatment were associated with immunologic responses to cART (Table 6).

TABLE 6 Association of virologic and immunologic responses 6 monthsafter starting cART with QXR QXR = 1 QXR < 1 P value Baseline QXRPatients with HIV-1 RNA <500 57 (68) 27 (32) 0.33* copies/mL at 6months, n (%) CD4^(†) cell increase [cells/ 82 (24 to 155) 40 (8 to 95)0.012^(†) pL] at 6 months, median (IQR) Follow-up QXR CD4^(†) cellincrease [cells/ 65 (29-110) 11 (0-35) 0.040^(†) μL] at 6 months, median(IQR) HIV-1 RNA viral loads at 6 months were available for 162/180patients with baseline QXR values. CD4 cell counts at 6 months availablefor 157/180 patients with baseline QXR values and for 58/70 withfollow-up QXR values. *Fisher's exact test; ^(†)Wilcoxon rank-sum test

Patients with baseline QXR<1 displayed significantly reduced CD4responses to cART as compared to those with QXR=1 (40 vs. 82 cells,P=0.012). This finding was also observed in patients with persistentviraemia and QXR<1 after 6 months of cART (11 vs. 65 cells, P=0.04). Thevirologic response to cART, defined here as suppression of HIV-1 RNAload to <500 copies/mL after 6 months of treatment, was not associatedwith QXR at baseline (P=0.33).

QXR and Viral Load are Strong Predictors of Clinical Progression

Kaplan-Meier estimates of the proportion of subjects who progressed to anew AIDS-defining illness or death, stratified according to QXR,revealed that QXR values strongly predicted the probability of diseaseprogression when measured before the initiation of cART (P=0.0002) (FIG.7) or, to a lesser extent, after 6 months of therapy in those with HIV-1loads>500 copies/mL (P=0.04).

To examine the independent effect of QXR<1 and X4-specific viral load ondisease progression, inventors applied Cox univariable and multivariableregression models (Table 7).

TABLE 7 Univariable and multivariable Cox proportional hazard regressionmodels of time from starting cART to new clinical AIDS defining illnessor death by using baseline QXR or baseline X4-specific load togetherwith CD4 cell counts and viral load as predictors.. MultivariableMultivariable HR, HR, Univariable HR including QXR including X4-specificVariable at baseline (95% CI) (95% o Cl) viral load (95% Cl)^(†) QXREqual 1 1.0 1.0 Less than 1 3.5 (1.8-67)  4.8 (2-3.10.0) X4-specificviral load, log10 copies/mL 0 (QXR = 1) 1.0 1.0 2.2-4.3 1.9 (0.9-4.3)3.7 (1.2-11.3) >4.3  7.1 (2.6-19.0) 5.9 (2.2-15.0) Doubling of CD4^(†)cell count  0.72 (0.60-0.88) 1.7 (1.0-3.0) 1.6 (0.84-2.9) Increase ofviral load by log₁₀ 2.2 (1.4-3.4) 1.7 (1.0-3.0) 1.6 (0.84-2.9) copies/mLHR: Hazard ratio; CI: Confidence interval * Multivariable model includesbaseline QXR and is adjusted for X4-specific and total viral load aswell as CD4^(†) cell count. ^(†)Multivariable model includes baselineX4-specific viral load, stratified according to the median of 61non-zero QXR values, and is adjusted for QXR, total viral load, andCD4^(†) cell count.

The adjusted multivariable hazard ratio (HR) for clinical progressionwas 4.8 (95% Cl: 2.3-10.0) for QXR<1 at baseline. For QXR<1 atfollow-up, the univariable HR was 3.7 (1.1-13.0); and of borderlinesignificance in the CD4 and HIV-1 RNA-adjusted multivariable model [HR2.9 (0.95-8.7), P=0.06].

X4-specific HIV-1 load was a similarly independent predictor, with HRsof 3.7 (1.2-11.3) for baseline X4-specific viral loads of 2.2-4.3 log₁₀copies/mL and 5.9 (2.2-15.0) for X4 loads>4.3 log₁₀ copies/mL.

Although total HIV-1 load and CD4 count were associated with clinicaldisease, QXR and X4-specific viral load strongly predicted diseaseprogression during cART, independent of and in addition to CD4 count ortotal viral load.

This example identifies HIV-1 coreceptor usage as a powerful predictorof response to cART. Patients harbouring X4 variants not only exhibiteda diminished immunologic response compared to those without X4 strains,but also displayed a markedly increased risk of progressing to AIDS ordeath despite treatment. The increased probability of clinicalprogression was observed in patients who displayed QXR<1 beforeinitiating cART and in those with persistent viraemia and QXR<1 after 6months of therapy.

Furthermore, patients with pretreatment X4-specific viral loads as lowas 2.2-4.3 log₁₀ copies/mLwere associated with a HR for clinicalprogression of 3.7, as compared to a HR of 1 for values<2.2 log10copies/mL. For X4 loads>4.3 log10 copies/mL, the HR was 5.9.

Because QXR and X4-specific viral load identifies a subset ofindividuals at increased risk of clinical progression, they promise tobe useful in clinical management. The quantification of QXR andX4-specific load may inform the decision to begin cART in untreatedpatients. It would be of interest to consider a clinical trialevaluating the initiation of cART in asymptomatic individuals withQXR<1, even those with CD4 counts>350 cells/uL. The aim of initiatingcART in such patients would be to shift the predominant viral populationfrom X4 to R5 (Philpott et al. (2001); Equils et al. (2000); and Skrabalet al. (2003)) as well as to reduce HIV-1 levels and thereby slowdisease progression.

Of the patients in this study who exhibited HIV-1 loads>500 copies/mLafter 6 months of cART, those harbouring X4 strains at follow-up were atincreased risk of disease progression compared with those displayingonly R5 variants. Therefore, patients with QXR<1 during cART mightbenefit from a change in therapy, with the aim of effective suppressionor reduction of X4 strains. Serial measurements of QXR and X4-specificviral load would permit quantitative monitoring of these markers.

This example also helps to elucidate the tremendous clinical success ofcART. Suppression of HIV-1 viraemia has become a major goal of treatmentbecause it has been associated with slower disease progression andprevention of drug resistance. A number of cohort studies have shownthat although many individuals initiating cART did not experiencesustained suppression of plasma viraemia (Ledergerber et al. (1999);Mezzaroma et al. (1999); Deeks et al. (2000); and Ledergerber et al.(2004), the majority of these patients derived significant immunologicand clinical benefits. In addition, studies have documented thatpatients with advanced HIV-1 disease who continued cART had a reducedmortality rate as compared to untreated individuals with comparable CD4counts and viral loads (Mocroft et al. (2003); Ledergerber et al.(2004); and Miller et al. (2004)). These reports demonstrate that cARTprovides clinical benefits beyond those mediated by the CD4 count andHIV-1 load.

Because cART has been shown to preferentially suppress X4 specific virusduring the first years of therapy, this data supports the idea that theclinical gains bestowed by treatment stem from two effects on HIV-1:suppression of viraemia and shift of the viral population from X4 towardR5-using virus. The finding that clinical response was related to QXR atfollow-up underscores this concept.

Previous analyses help to explain how X4 variants may affect responsesto cART (Blaak et al. (2000); Kreisberg et al. (2001); and Jekle et al.(2003). The cytopathicity of HIV-1 primary isolates depends uponcoreceptor usage and not the patient's disease status (Kreisberg et al.(2001)). One report focused on HIV-1 isolates from patients withpersistent viraemia and drug resistance during cART, and comparedcharacteristics of viruses from patients exhibiting a CD4 countincrease, called a “paradoxical response,” to those without animmunologic response (Solomon et al. (2005) J. Acquir. Immune Defic.Syndr. 40:140-8). Viral variants from the non-responders were morelikely to demonstrate high replicative capacity, induce apoptosis, anduse the X4 coreceptor than those from the responders.

Studies of paradoxical responders have suggested that the benefits ofcART may stem from partial suppression of HIV-1 load in these patientsand the diminished replicative capacity exhibited by many drug-resistantviruses (Deeks et al. (2000) J. Infect. Dis. 181:946-53). This reportsupports the role of preferential suppression of X4 variants as anadditional means by which cART may lead to CD4 cell reconstitution orstability without complete viral suppression. The relationship betweenQXR and response to cART carries important implications for research onpathogenesis and therapeutics as well as clinical care.

A quantitative HTA permitted us to link clinical disease progression toQXR and X4-specific viral load. This sensitive assay revealed that >50%of the samples in this study harbouring X4 variants displayed QXR≧0.75,indicating that X4 strains comprised <25% of their viral quasispecies.These data demonstrate that the presence of X4 strains was associatedwith an increased probability of disease progression even when suchvariants comprised a small proportion of the HIV-1 population.

Because this investigation focused on a subset of SHCS participants whoinitiated cART in 1995-1998 (Ledergerber et al. (1999)), our selectionof patients relied on the availability of cryopreserved plasma samples.Although the patients inventors studied were demographically comparableto the entire SHCS population, the non-progressors described in thisreport displayed more advanced immunosuppression than non-progressorswhose samples were unavailable.

There is no obvious explanation for this unintentional imbalance.Because it diminishes the difference between progressors andnon-progressors, inventors are confident, however, that the findings ofthis study remain valid. In addition, fewer samples were available forQXR analysis at follow-up than at baseline, owing primarily to theeffectiveness of cART in suppressing HIV-1 load to <500 copies/mL.

The invention is further described by the following numbered paragraphs:

1. A diagnostic method comprising determining the viral load of apopulation of acquired immunodeficiency (AIDS) virus using the CXCR4coreceptor (X4-specific viral load) in a patient-derived biologicalsample comprising the steps of:

-   -   (a) screening individual molecular clones of patient-derived        acquired immunodeficiency primary isolate with a heteroduplex        tracking assay to determine CCR5 coreceptor usage and CXCR4        coreceptor usage of each individual molecular clone;    -   (b) determining the proportion of HIV using the CCR5 coreceptor        (R5) versus the CXCR4 coreceptor (X4) wherein the proportion is        expressed as a variable called the Quantity of X4 and R5 (QXR),        which represents the fraction of virus in a specimen using the        R5 coreceptor;    -   (c) determining coreceptor specific viral loads of the        patient-derived acquired immunodeficiency primary isolate        wherein the R5-specific viral load=(VL)(QXR) and the X4-specific        viral load=(VL)(1-QXR        wherein initiation or change of antiretroviral therapy may be        considered anytime that the X4-specific viral load is greater        than zero.        2. The diagnostic method according to paragraph 1, wherein if        QXR=1, almost all of the viruses in the population use the R5        coreceptor;

further wherein if QXR=0, almost all of the viruses in the populationuse the X4 coreceptor;

further wherein if QXR<1, the viruses in the population use a mixture ofthe R5 and X4 coreceptors.

3. The diagnostic method according to paragraph 1, wherein thebiological sample is any bodily fluid or tissue.4. The diagnostic method according to paragraph 3, wherein thebiological sample is a bodily fluid selected from the group consistingof blood, plasma, and spinal fluid.5. The diagnostic method according to paragraph 1, wherein theindividual molecular clones each comprise a DNA sequence correspondingto a portion of the HIV genome, the DNA sequence comprising at least aportion of the genetic determinates of coreceptor usage.6. The diagnostic method according to paragraph 5, wherein the geneticdeterminates are derived from the env gene.7. The diagnostic method according to paragraph 1, wherein the molecularclones each are derived from RNA of the patient-derived HIV andcorrespond to the HIV genome or a portion thereof and which comprise thegenetic determinates of coreceptor usage or a portion thereof.8. The diagnostic method according to paragraph 7, wherein the molecularclones are prepared by PCR of the RNA of the patient-derived HIV and atleast one set of oligonucleotide primers.9. The diagnostic method according to paragraph 8, wherein at least oneset of oligonucleotide primers consists of the first set of primers inTable 3.10. The diagnostic method according to paragraph 8, wherein the at leastone set of oligonucleotide primers includes a second set ofoligonucleotide primers, the second set consisting of the second set ofprimers in Table 3.11. The diagnostic method according to paragraph 1, wherein the numberof individual molecular clones is at least 20.12. The diagnostic method according to paragraph 1, wherein theheteroduplex tracking assay comprises the steps of:

-   -   (a) amplifying the individual molecular clone or a portion        thereof by PCR to provide amplified DNA comprising the genetic        determinates of coreceptor usage or a portion thereof;    -   (b) forming a population of heteroduplex molecules by contacting        the amplified DNA with a labeled probe complementary to the        amplified DNA under conditions sufficient to form        heteroduplexes;    -   (c) separating the population of heteroduplex molecules using a        separation means;    -   (d) detecting the presence or absence of heteroduplex molecules;        wherein the presence or absence of heteroduplex molecules        reveals coreceptor usage.        13. The diagnostic method according to paragraph 12, wherein the        labeled probe is derived from a known HIV-1 CCR5 clone.        14. The diagnostic method according to paragraph 12, wherein the        labeled probe is derived from a known HIV-1 CXCR4 clone.        15. The diagnostic method according to paragraph 12, wherein the        labeled probe comprises a detectable moiety, a radioisotope,        biotin, a fluorescent moiety, a fluorophore, a chemiluminescent        moiety, or an enzymatic moiety.        16. The diagnostic method according to paragraph 1, wherein the        method is used (a) to assess or predict the degree of HIV        progression, (b) to determine when to start or change        antiretroviral treatment, or (c) to monitor the efficacy of        antiretroviral treatment.        17. The diagnostic method according to paragraph 2, wherein the        method is used (a) to assess or predict the degree of HIV        progression, (b) to determine when to start or change        antiretroviral treatment, or (c) to monitor the efficacy of        antiretroviral treatment.        18. A method of determining when to initiate antiretroviral        therapy in a patient comprising determining the viral load of a        population of AIDS virus using the CXCR4 coreceptor (X4-specific        viral load) in a patient-derived biological sample comprising        the steps of:    -   (a) screening individual molecular clones of patient-derived        acquired immunodeficiency primary isolate with a heteroduplex        tracking assay to determine the CCR5 coreceptor usage and the        CXCR4 coreceptor usage of each individual molecular clone;    -   (b) determining the proportion of HIV using the CCR5 coreceptor        (R5) versus the CXCR4 coreceptor (R4) wherein the proportion is        expressed as a variable called the Quantity of X4 and R5 (QXR),        which represents the fraction of virus in a specimen using the        R5 coreceptor;    -   (c) determining coreceptor specific viral loads of the        patient-derived acquired immunodeficiency primary isolate        wherein the R5-specific viral load=(VL)(QXR) and the X4-specific        viral load=(VL)(1−QXR),        wherein initiation or change of antiretroviral therapy may be        considered anytime that the X4-specific viral load is greater        than zero.        19. The method according to paragraph 18, wherein if QXR=1,        almost all of the viruses in the population use the R5        coreceptor;

further wherein if QXR=0, almost all of the viruses in the populationuse the X4 coreceptor;

further wherein if QXR<1, the viruses in the population use a mixture ofthe R5 and X4 coreceptors.

20. The method according to paragraph 18, wherein the biological sampleis a bodily fluid selected from the group consisting of blood, plasma,and spinal fluid.21. The method according to paragraph 18, wherein the individualmolecular clones each comprise a DNA sequence corresponding to a portionof the HIV genome, the DNA sequence comprising at least a portion of thegenetic determinates of coreceptor usage.22. The method according to paragraph 21, wherein the geneticdeterminates are derived from the env gene.23. The method according to paragraph 18, wherein the molecular cloneseach are derived from RNA of the patient-derived HIV and correspond tothe HIV genome or a portion thereof and which comprise the geneticdeterminates of coreceptor usage or a portion thereof.24. The method according to paragraph 23, wherein the molecular clonesare prepared by RT-PCR of the RNA of the patient-derived HIV and atleast one set of oligonucleotide primers.25. The method according to paragraph 24, wherein at least one set ofoligonucleotide primers consists of the first set of primers in Table 3.26. The method according to paragraph 24, wherein the at least one setof oligonucleotide primers includes a second set of oligonucleotideprimers, the second set consisting of the second set of primers in Table3.27. The method according to paragraph 18, wherein the number ofindividual molecular clones is at least 20.28. The method according to paragraph 18, wherein the heteroduplextracking assay comprises the steps of:

-   -   (a) amplifying the individual molecular clone or a portion        thereof by PCR to provide amplified DNA comprising the genetic        determinates of coreceptor usage or a portion thereof;    -   (b) forming a population of heteroduplex molecules by contacting        the amplified DNA with a labeled probe complementary to the        amplified DNA under conditions sufficient to form        heteroduplexes;    -   (c) separating the population of heteroduplex molecules using a        separation means;    -   (d) detecting the presence or absence of heteroduplex molecules;        wherein the presence or absence of heteroduplex molecules        reveals coreceptor usage.        29. The method according to paragraph 28, wherein the labeled        probe is derived from a known HIV-1 CCR5 clone.        30. The method according to paragraph 28, wherein the labeled        probe is derived from a known HIV-1 CXCR4 clone.        31. The method according to paragraph 28, wherein the labeled        probe comprises a detectable moiety, a radioisotope, biotin, a        fluorescent moiety, a fluorophore, a chemiluminescent moiety, or        an enzymatic moiety.        32. The method according to paragraph 18, wherein the        antiretroviral therapy is any suitable antiretroviral treatment        regimen.        33. The method according to paragraph 32, wherein the        antiretroviral therapy is selected from the group consisting of        combination antiretroviral therapy (cART), protease inhibitors,        fusion inhibitors, integrase inhibitors, coreceptor specific        agents, nonnucleoside analogue reverse transcriptase inhibitors        and nucleoside analogue reverse transcriptase inhibitors.        34. The method according to paragraph 33, wherein the nucleoside        analogue reverse transcriptase inhibitor is 3TC.        35. The method according to paragraph 33, wherein the nucleoside        analogue reverse transcriptase inhibitor is AZT.        36. The method according to paragraph 33, wherein the        nonnucleoside analogue reverse transcriptase inhibitor is        nevirapine.        37. A method of monitoring the efficacy of antiretroviral        therapy in a patient comprising determining the viral load of a        population of AIDS virus using the CXCR4 coreceptor (X4-specific        viral load) in a patient-derived biological sample comprising        the steps of:    -   (a) screening individual molecular clones of patient-derived        acquired immunodeficiency primary isolate with a heteroduplex        tracking assay to determine the CCR5 coreceptor usage and the        CXCR4 coreceptor usage of each individual molecular clone;    -   (b) determining the proportion of HIV using the CCR5 coreceptor        (R5) versus the CXCR4 coreceptor (R4) wherein the proportion is        expressed as a variable called the Quantity of X4 and R5 (QXR),        which represents the fraction of virus in a specimen using the        R5 coreceptor;    -   (c) determining coreceptor specific viral loads of the        patient-derived acquired immunodeficiency primary isolate        wherein the R5-specific viral load=(VL)(QXR) and the X4-specific        viral load=(VL)(1−QXR);        wherein X4-specific viral load strongly predicts disease        progression during cART.        38. The method according to paragraph 37, wherein if QXR=1,        almost all of the viruses in the population use the R5        coreceptor;

further wherein if QXR=0, almost all of the viruses in the populationuse the X4 coreceptor;

further wherein if QXR<1, the viruses in the population use a mixture ofthe R5 and X4 coreceptors.

39. The method according to paragraph 37, wherein the biological sampleis a bodily fluid, such as blood, plasma, and spinal fluid.40. The method according to paragraph 37, wherein the individualmolecular clones each comprise a DNA sequence corresponding to a portionof the HIV genome, the DNA sequence comprising at least a portion of thegenetic determinates of coreceptor usage.41. The method according to paragraph 40, wherein the geneticdeterminates are derived from the env gene.42. The method according to paragraph 37, wherein the molecular cloneseach are derived from RNA of the patient-derived HIV and correspond tothe HIV genome or a portion thereof and which comprise the geneticdeterminates of coreceptor usage or a portion thereof.43. The method according to paragraph 42, wherein the molecular clonesare prepared by RT-PCR of the RNA of the patient-derived HIV and atleast one set of oligonucleotide primers.44. The method according to paragraph 43, wherein at least one set ofoligonucleotide primers consists of the first set of primers in Table 3.45. The method according to paragraph 43, wherein the at least one setof oligonucleotide primers includes a second set of oligonucleotideprimers, the second set consisting of the second set of primers in Table3.46. The method according to paragraph 37, wherein the number ofindividual molecular clones is at least 20.47. The method according to paragraph 37, wherein the heteroduplextracking assay comprises the steps of:

-   -   (a) amplifying the individual molecular clone or a portion        thereof by PCR to provide amplified DNA comprising the genetic        determinates of coreceptor usage or a portion thereof;    -   (b) forming a population of heteroduplex molecules by contacting        the amplified DNA with a labeled probe complementary to the        amplified DNA under conditions sufficient to form        heteroduplexes;    -   (c) separating the population of heteroduplex molecules using a        separation means;    -   (d) detecting the presence or absence of heteroduplex molecules;        wherein the presence or absence of heteroduplex molecules        reveals coreceptor usage.        48. The method according to paragraph 47, wherein the labeled        probe is derived from a known HIV-1 CCR5 clone.        49. The method according to paragraph 47, wherein the labeled        probe is derived from a known HIV-1 CXCR4 clone.        50. The method according to paragraph 47, wherein the labeled        probe comprises a detectable moiety, a radioisotope, biotin, a        fluorescent moiety, a fluorophore, a chemiluminescent moiety, or        an enzymatic moiety.        51. The method according to paragraph 37, wherein the        antiretroviral therapy is any suitable antiretroviral treatment        regimen.        52. The method according to paragraph 51, wherein the        antiretroviral therapy is selected from the group consisting of        combination antiretroviral therapy (cART), protease inhibitors,        fusion inhibitors, integrase inhibitors, coreceptor specific        agents, nonnucleoside analogue reverse transcriptase inhibitors        and nucleoside analogue reverse transcriptase inhibitors.        53. The method according to paragraph 52, wherein the nucleoside        analogue reverse transcriptase inhibitor is 3TC.        54. The method according to paragraph 52, wherein the nucleoside        analogue reverse transcriptase inhibitor is AZT.        55. The method according to paragraph 52, wherein the        nonnucleoside analogue reverse transcriptase inhibitor is        nevirapine.        56. A diagnostic method for determining the viral load of a        population of acquired immunodeficiency virus using the CXCR4        coreceptor (X4-specific viral load) in a patient-derived        biological sample.        57. A diagnostic method comprising determining the viral load of        a population of acquired immunodeficiency (AIDS) virus using the        CXCR4 coreceptor (X4-specific viral load) in a patient-derived        biological sample comprising the steps of:    -   (a) screening individual molecular clones of patient-derived        acquired immunodeficiency primary isolate with a V3 loop        sequencing assay to determine CCR5 coreceptor usage and CXCR4        coreceptor usage of each individual molecular clone;    -   (b) determining the proportion of HIV using the CCR5 coreceptor        (R5) versus the CXCR4 coreceptor (X4) wherein the proportion is        expressed as a variable called the Quantity of X4 and R5 (QXR),        which represents the fraction of virus in a specimen using the        R5 coreceptor;    -   (c) determining coreceptor specific viral loads of the        patient-derived acquired immunodeficiency primary isolate        wherein the R5-specific viral load=(VL)(QXR) and the X4-specific        viral load=(VL)(1−QXR).        58. The diagnostic method according to paragraph 57, wherein the        V3 loop sequencing assay is a heteroduplex tracking assay.        59. The diagnostic method according to paragraph 58, further        comprising an ultra deep sequencing assay.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

1-4. (canceled)
 5. A diagnostic method comprising determining the viralload of a population of acquired immunodeficiency (AIDS) virus using theCXCR4 coreceptor (X4-specific viral load) in a patient-derivedbiological sample comprising the steps of: (a) screening individualmolecular clones of patient-derived acquired immunodeficiency primaryisolate with a heteroduplex tracking assay to determine CCR5 coreceptorusage and CXCR4 coreceptor usage of each individual molecular clone; (b)determining the proportion of HIV using the CCR5 coreceptor (R5) versusthe CXCR4 coreceptor (X4) wherein the proportion is expressed as avariable called the Quantity of X4 and R5 (QXR), which represents thefraction of virus in a specimen using the R5 coreceptor; (c) determiningcoreceptor specific viral loads of the patient-derived acquiredimmunodeficiency primary isolate wherein the R5-specific viralload=(VL)(QXR) and the X4-specific viral load=(VL)(1−QXR).
 6. Thediagnostic method according to claim 5, wherein the biological sample isany bodily fluid or tissue.
 7. The diagnostic method according to claim6, wherein the biological sample is a bodily fluid selected from thegroup consisting of blood, plasma, and spinal fluid.
 8. The diagnosticmethod according to claim 5, wherein the individual molecular cloneseach comprise a DNA sequence corresponding to a portion of the HIVgenome, the DNA sequence comprising at least a portion of the geneticdeterminates of coreceptor usage.
 9. The diagnostic method according toclaim 6, wherein the genetic determinates are derived from the env gene.10. The diagnostic method according to claim 5, wherein the molecularclones each are derived from RNA of the patient-derived HIV andcorrespond to the HIV genome or a portion thereof and which comprise thegenetic determinates of coreceptor usage or a portion thereof.
 11. Thediagnostic method according to claim 10, wherein the molecular clonesare prepared by RT-PCR of the RNA of the patient-derived HIV and atleast one set of oligonucleotide primers.
 12. The diagnostic methodaccording to claim 11, wherein at least one set of oligonucleotideprimers consists of the first set of primers in Table
 3. 13. Thediagnostic method according to claim 11, wherein the at least one set ofoligonucleotide primers includes a second set of oligonucleotideprimers, the second set consisting of the second set of primers in Table3.
 14. The diagnostic method according to claim 5, wherein the number ofindividual molecular clones is at least
 20. 15. The diagnostic methodaccording to claim 5, wherein the heteroduplex tracking assay comprisesthe steps of: (a) amplifying the individual molecular clone or a portionthereof by PCR to provide amplified DNA comprising the geneticdeterminates of coreceptor usage or a portion thereof; (b) forming apopulation of heteroduplex molecules by contacting the amplified DNAwith a labeled probe complementary to the amplified DNA under conditionssufficient to form heteroduplexes; (c) separating the population ofheteroduplex molecules using a separation means; (d) detecting thepresence or absence of heteroduplex molecules; wherein the presence orabsence of heteroduplex molecules reveals coreceptor usage.
 16. Thediagnostic method according to claim 15, wherein the labeled probe isderived from a known HIV-1 CCR5 clone.
 17. The diagnostic methodaccording to claim 15, wherein the labeled probe is derived from a knownHIV-1 CXCR4 clone.
 18. The diagnostic method according to claim 15,wherein the labeled probe comprises a detectable moiety, a radioisotope,biotin, a fluorescent moiety, a fluorophore, a chemiluminescent moiety,or an enzymatic moiety.
 19. The diagnostic method according to claim 5,wherein the method is used (a) to assess or predict the degree of HIVprogression, (b) to determine when to start or change antiretroviraltreatment, or (c) to monitor the efficacy of antiretroviral treatment.20. (canceled)
 21. A method of determining when to initiateantiretroviral therapy in a patient comprising determining the viralload of a population of AIDS virus using the CXCR4 coreceptor(X4-specific viral load) in a patient-derived biological samplecomprising the steps of: (a) screening individual molecular clones ofpatient-derived acquired immunodeficiency primary isolate with aheteroduplex tracking assay to determine the CCR5 coreceptor usage andthe CXCR4 coreceptor usage of each individual molecular clone; (b)determining the proportion of HIV using the CCR5 coreceptor (R5) versusthe CXCR4 coreceptor (R4) wherein the proportion is expressed as avariable called the Quantity of X4 and R5 (QXR), which represents thefraction of virus in a specimen using the R5 coreceptor; (c) determiningcoreceptor specific viral loads of the patient-derived acquiredimmunodeficiency primary isolate wherein the R5-specific viralload=(VL)(QXR) and the X4-specific viral load=(VL)(1−QXR); whereinantiretroviral therapy is initiated anytime that the X4-specific viralload is greater than zero.
 22. A method of monitoring the efficacy ofantiretroviral therapy in a patient comprising determining the viralload of a population of AIDS virus using the CXCR4 coreceptor(X4-specific viral load) in a patient-derived biological samplecomprising the steps of: (a) screening individual molecular clones ofpatient-derived acquired immunodeficiency primary isolate with aheteroduplex tracking assay to determine the CCR5 coreceptor usage andthe CXCR4 coreceptor usage of each individual molecular clone; (b)determining the proportion of HIV using the CCR5 coreceptor (R5) versusthe CXCR4 coreceptor (R4) wherein the proportion is expressed as avariable called the Quantity of X4 and R5 (QXR), which represents thefraction of virus in a specimen using the R5 coreceptor; (c) determiningcoreceptor specific viral loads of the patient-derived acquiredimmunodeficiency primary isolate wherein the R5-specific viralload=(VL)(QXR) and the X4-specific viral load=(VL)(1−QXR) whereinX4-specific viral load strongly predicts disease progression duringcART.